Website: www.fermentor.co.in

Food Beverages Textile Leather Agriculture Bio Pharma

Effluent Treatment Enzyme :

INTRODUCTIONProtecting ecology is our duty. We are thus protecting our future generation. Waste water treatment has assumed great significance intoday’s context where protecting the environment is a prime concern. The main objective of waste water treatment is to treat the effluentbefore it is discharged so that the environment is not polluted. Waste water treatment in general refers to treatment of suspended andfloatable material, treatment of biodegradable organics and the elimination of pathogenic organisms. The contaminants in waste water areremoved by physical chemical and biological means. These organisms are effectively removed by an enzyme called ENVIRO NZYME.

ROLE OF AN ENZYME?

An enzyme is a chemical catalyst that breaks up long, complex waste molecules (Hydrolytic reaction) into smaller pieces, which can then bedigested directly by the bacteria. Enzymes are manufactured and used by bacteria in order to digest waste. ENVIRO NZYME – G a dry freeflowing powder is a concentrated source of hydrolytic enzymes and eight strains of natural bacteria that are genetically capable of producingconcentrations of enzymes in waste treatment systems under aerobic and anaerobic conditions. ENVIRO NZYME –G is used for reducing theBOD, COD levels as well as reducing the sludge volumes odour and colour in the effluent and sewage treatment plants.

APPLICATION:

This product finds application in industries like

Agro Breweries & Distilleries Coffee & Tea estates Dairy

Food Processing Hotel Leather Wheat gluten Paper Pharmaceutical Sugar Textiles Wonderzyme Municipal & Industrial waste treatment.

WHERE THE ENZYME TO BE USED?

The areas in which the ENVIRO-NZYME-G, is used are:

Oxidation Lagoons Sludge Pits Grease Traps Aeration Tanks Imhoff Tanks Digestors(Aerobic & Anaerobic) Septic Tanks Clarifies Trickling Filters Collection \systems

WHAT DOES ENZYME DO?

ENVIRO NZYME-G,when added helps to

Remove odour Sludge volume will be reduced more, as more solids are digested. Sludge will be easier to pump, process and dewater, and will have less odour too. The capacity of the system will be effectively increased, because more waste can be processed, more efficiently,in lesser time. The whole system will be able to absorb the shock of toxic influent. Easier to balance the treatment system. Bacterial Oxidation of the liquid phase will be faster and more complete Digesters will operate evenly and uniformly, easier planning and routine waste. Effluent water standards will be met more consistently, helping to maintain a clean environment and safe drinking water for the

population.

DOSAGE

Dosage rate will vary with retention times, BOD/COD ranges, water temperature, pH, sludge builtup and pond depth. Initial treatment of anywaste system should be eight times the volume of ENVIRO NZYME-G. Normal preventive maintenance* in oxidation, lagoons, digesters,sludge pits, clarifiers, imhoff tanks and collection system is 2ppm of ENVIRO NZYME-G per day. The 2ppm is determined by the total weightof the waste water in the system.

For one lakh litres of effluent, use 200 gms of enzyme per day.

The normal preventive maintenance rates are based on the following criteria. Minimum retention time of 5 days. BOD ranges of 1150 to 250 ppm. pH of 6.5 to 7.5

Note:

If the product is to be used in a waste treatment system with almost continuous flow and with hydraulic retention of less than 24 hrs,the dryproducts may be soaked in luke warm water at 30C for 8 hrs in 1% water solution to fully activate the biological components of the productsand to allow the bacteria to get a quick start when introduced into the system. Soaking period of over 10-12 hrs are recommended.

In multiple – level applications, it is recommended that ENVIRO NZYME-G be applied at the lowest level first. Start at the bottom or end ofthe system from floor to floor or from section as appropriate. The reason for this is that where ENVIRO NZYME-G to be applied at the top orbeginning of the system, the waste material washed away might cause problems in the lower portion of the system unless this portion isalready cleared. During normal preventive maintenance, the point of application should be in an area which allows the product the max. timein the system.

HOW TO ADD THE ENVIRO NZYME-G INTO THE SYSTEM?

ENVIRO NZYME-G should be activated by mixing them in a bucket of warm water (not hot water) for few minutes. Pour the slurry into thesystem at a point where normal water flow will disperse it evenly. The best way is to pour it into the waste stream inside the plant, or justbefore it flows out into the waste stream inside the plant, or just before it flows out into the pond. If this is not possible, pour it directly intothe middle of the pond, or around the perimeter.

WHEN SHALL THE RESULTS BE ACHIEVED?

Bad odours and sludge accumulations take wonths and years to build up, and it will take some time to get rid of them. Begin the treatmentwith double the normal weekly dosage for a period of 3 to 4 weeks. After this start up period, one will begin to see significant improvements,and can cut back to normal weeklydose. Severe accumulation of sludge may take one or more years to get eliminated.

WHY TO HAVE A CONTINUOUS TREATMENT?

The bacteria in these products are very efficient at digesting waste-many time better then the natural bacteria. However, they are not as“strong” as the natural bacteria. If the treatment is stopped, the natural occurring bacteris are undesirable because they work slowly,produce odours and can be pathogenic.

WHICH OF THE DISINFECTANT CAN KILL BACTERIA?

Solutions: Do not chlorinate frequently. On application increase the dosage of ENVIRO NZYME-G.

OPERATIONAL PARAMETERS

ENVIRO NZYME-G contains a complex of bacteria and enzymes which perform in an adqueous solution within the following parameters:

pH

optimum 7.0 minimum 5.0 maximum 9.0 (Should not be too acidic)

Dissolved Oxygen

optimum 2ppm minimum 1 ppm

Carbon/Nitrogen

Ratio(C/N) Optimum 10:1 Maximum20:1

Temperature

optimum 30C minimum 10C maximum 40C

Negative effects on the biological growth rates are possible in the presence4 of varying concentrations of heavy metal in theadeeous solution (less than 20 ppm)

Residence time or the time biology needs to acclimatize to break down the organic waste is 5 days.

PRODUCT SPECIFICATION

BACTERIA COUNT

Aerobic BacteriaAnaerobic BacteriaFacultative BacteriaTotal plate CountE.ColiSalmonellaToxins

Min 1.5 billion colonies/gmMin 2.5 billion colonies/gmMin 2.0 billion colonies/gmMin 6.0 billion colonies/gmAbsent in 0.1 gmAbsent in 25 gmAbsent in 0.1 gm

APPEARANCE

Medium tan, free flowing granular powder with a yeasty odour.

STORAGE AND HANDLING

For a longer Shelf life keep in a dry storage area under 38C. Shelf life under these conditions estimated to be in excess of one year.

Temperature: More effective in our tropical lands

Avoid inhalation of dust and eye contact. Not for food use nor for contact with food preparatory surfaces o equipment.

Enzyme Products & SolutionsCollapse All | Expand All

Foodo Bakeryo Ice Cream Beverageso Breweryo Juiceo Wineo Starcho Sugar Textile Leather Agricultureo Animal Feedo Agriculture Growth Enzyme Bio Pharma Waste water solutionso Effluent water treatmento Detergent

Food :

Our team of dedicated researchers constantly strives on innovating newer products to help in maximizing output while enhancing quality.Our solutions provide many advantages, including higher quality products, energy efficiency, and a safer working environment. Processingequipment also lasts longer since the milder conditions reduce corrosion.

Bakery :

With an increasing consumer desire for natural products and better mouth feel,food manufacturers should use enzymes or yeast instead ofadditives to improve their products.Enzymes have a multitude of benefits.From the consumer angle they can extend shelf life of goods from three to 10-14 days, dependent onthe product and recipe.They have the ability to strengthen the stability of the dough. During proofing and transportation on belts inside thebakery dough should not collapse due to the mechanical stress and you can apply enzymes to give it certain strength.Enzymes also help tocorrect the flour to give it a consistent baking performance, independent of the fluctuating flour quality.

Bakery Solutions:

Glucose Oxidase Xylanase Alpha Amylase DIACETYL TARTARIC ACID ESTER OF MONO-DIGLYCERIDES (DATEM) SODIUM STEAROYL LACTYLATE (SSL) CRACKER-ZYME (Biscuit Enzyme) COOKIZYME P Wheat gluten SOYA FLOUR PAN RELEASE P.G.P.R. Puffzyme Wonderzyme Whey Protein PAPAIN

Ice creams :Ice cream enzymes provide the best solution for our consumers. They help reduce ice crystals and enhance the flavor of the product.Resulting in richer & creamer ice cream.

Ice Cream Solutions:

Cremozyme series Softy mix Thickzyme

Beverages :

Enzymes are the key to maintaining sustained high quality in the beverage indutries. Revolutionary solutions are your key to cost savings,increased outputs, and consistent quality. We at EIPL, constantly create and innovate solutions that combine profitability with sustainabilityhelping your business to grow.

Brewery :

Profit from the power of enzymes and sharpen your competitive edge with cost-effective production, optimal raw material use and higheryield. That is why our brewery solutions focuses on increasing performance with local raw materials; helps in boosting quality; aids inincreasing consistency in mashing process and adjunct cooking; whileimproving mash separation and beer filtration with increasedfermentabilityand controlled attenuation. Whether it is achieving consistent attenuation or developing a brand extension with light and low-calorie claims, our team of experts is ready to help and recommend required solutions in achieving your desired goals.

Brewery Solutions:

THERMO-NZYME L GLUCO-NZYME L Winezyme Finezyme – beer filteration agent PRO EXL – chill proofing agent for beer PAPAIN

Wine :Enzymes have been used, though unknowingly, for thousands of years in the production of various food products. Today, commerciallyproduced enzymes benefit a number of industries, including the wine industry. Commercial enzymes for wine are used in four differentapplications in winemaking: maceration and extraction, clarification, maturation, and filtration.

Wine Solutions:

Winezyme

Juices :The use of enzymes in juice processing helps assure that the maximum amount of juice is removed from the fruit, thereby reducing wasteand controlling costs.

Our broad range of solutions benefits the juice industry by easing the production of healthy, tangy juices.

Juices Solutions:

Tropizyme P

Starch Solutions:

Thermo stable alpha amylase

Sugar Solutions:

Dextranase Invertase

Textile :

Textile processing has benefited greatly on both environmental and product quality aspects through the use of enzymes. Our productbenefits the environment by helping in lower discharge of chemicals and wastewater and decreased handling of hazardous chemicals fortextile workers.

Through innovative enzyme solutions, EIPL helps you gain competitive advantages and make use of unique opportunities to stay ahead ofindustry trends. That is how we contribute to creating a better industry and world.

DENI-SIZE (Desizing Enzyme) DENI-WASH BP (Biopolishing with High Fading) DENI-WASH BPL (Biopolishing with low fading) DENI-WASH RL (Acid Cellulase for Denims) Anti-back staining agents DENI-WASH VT (Neutral Cellulase for Denims) PUMICE STONES

Leather :

Turning raw leather into finished goods involves multiple processes in which manufacturers attempt to optimize quality and yield whilereducing costs and environmental pollution.

With enzyme-assisted leather industrial solutions, it is possible to reduce the chemical requirements and obtain a cleaner product and ahigher area yield with fewer chemicals in the wastewater. In processes such as Dehairing;since the enzyme does not dissolve the hair as thechemicals do, it is possible to filter out the hair, thus reducing the chemical and biological oxygen demand of the wastewater. Thusbenefiting the environment by lowering the chemical load in wastewaterand reducing odor.

BIOBATE AC (Acid Bating Enzyme) Biobate AL (alkaline Bating Enzyme) Soakzyme Micrograin (Anti-wrinkle And Unhairing Enzyme Pancreatin (Industrial)

Agriculture :

According to researchers, demand for agricultural output is projected to grow by at least 75% by 2050. This means that farmers will need toproduce more food in the years to come, than they have. People are increasingly becoming aware of sustainable farming practices as thekey to meeting the world’s agricultural demands far into the future.

Not only do our agricultural solutions help ensure that our customers remain competitive, they also ensure that modern farming practicesare sophisticated enough to provide for future generations.

Animal nutrition :

Enzymes for animal nutrition help improve digestion processes and make the most of the feed while keeping feed costs low.

Swine and poultry are the most interesting types of animals when it comes to adding enzymes to feed because their digestive systems donot produce enzymes capable of breaking down plant cell walls. The digestive tract has to break down the feed into a form that the animalscan absorb and utilize. This way, the nutritional value inherent in the feed becomes accessible to the animal.

While enzymes ensure greater efficiency in the production of animal products, including meat and eggs, adding enzymes to feed alsominimizes the environmental impact of animal production.

Crop production :

Enzymatic pesticides and microbial yield and fertility enhancers help farmers to enjoy healthier crops and higher yields, with some solutionsimproving the availability and prudent management of nonrenewable phosphate fertilizer.

Agriculture Solutions:

Animal Feedo Poultry and animal feed

Agriculture Growth Enzymeo Agro Boostero Earthzyme

Effluent Treatment Enzyme :

INTRODUCTIONProtecting ecology is our duty. We are thus protecting our future generation. Waste water treatment has assumed great significance intoday’s context where protecting the environment is a prime concern. The main objective of waste water treatment is to treat the effluentbefore it is discharged so that the environment is not polluted. Waste water treatment in general refers to treatment of suspended andfloatable material, treatment of biodegradable organics and the elimination of pathogenic organisms. The contaminants in waste water areremoved by physical chemical and biological means. These organisms are effectively removed by an enzyme called ENVIRO NZYME.

ROLE OF AN ENZYME?

An enzyme is a chemical catalyst that breaks up long, complex waste molecules (Hydrolytic reaction) into smaller pieces, which can then bedigested directly by the bacteria. Enzymes are manufactured and used by bacteria in order to digest waste. ENVIRO NZYME – G a dry freeflowing powder is a concentrated source of hydrolytic enzymes and eight strains of natural bacteria that are genetically capable of producingconcentrations of enzymes in waste treatment systems under aerobic and anaerobic conditions. ENVIRO NZYME –G is used for reducing theBOD, COD levels as well as reducing the sludge volumes odour and colour in the effluent and sewage treatment plants.

APPLICATION:

This product finds application in industries like

Agro Breweries & Distilleries Coffee & Tea estates Dairy Food Processing Hotel Leather Wheat gluten Paper Pharmaceutical Sugar Textiles Wonderzyme Municipal & Industrial waste treatment.

WHERE THE ENZYME TO BE USED?

The areas in which the ENVIRO-NZYME-G, is used are:

Oxidation Lagoons Sludge Pits Grease Traps Aeration Tanks Imhoff Tanks Digestors(Aerobic & Anaerobic) Septic Tanks Clarifies Trickling Filters Collection \systems

WHAT DOES ENZYME DO?

ENVIRO NZYME-G,when added helps to

Remove odour Sludge volume will be reduced more, as more solids are digested. Sludge will be easier to pump, process and dewater, and will have less odour too. The capacity of the system will be effectively increased, because more waste can be processed, more efficiently,in lesser time. The whole system will be able to absorb the shock of toxic influent. Easier to balance the treatment system. Bacterial Oxidation of the liquid phase will be faster and more complete Digesters will operate evenly and uniformly, easier planning and routine waste. Effluent water standards will be met more consistently, helping to maintain a clean environment and safe drinking water for the

population.

DOSAGE

Dosage rate will vary with retention times, BOD/COD ranges, water temperature, pH, sludge builtup and pond depth. Initial treatment of anywaste system should be eight times the volume of ENVIRO NZYME-G. Normal preventive maintenance* in oxidation, lagoons, digesters,sludge pits, clarifiers, imhoff tanks and collection system is 2ppm of ENVIRO NZYME-G per day. The 2ppm is determined by the total weightof the waste water in the system.

For one lakh litres of effluent, use 200 gms of enzyme per day.

The normal preventive maintenance rates are based on the following criteria. Minimum retention time of 5 days. BOD ranges of 1150 to 250 ppm. pH of 6.5 to 7.5

Note:

If the product is to be used in a waste treatment system with almost continuous flow and with hydraulic retention of less than 24 hrs,the dryproducts may be soaked in luke warm water at 30C for 8 hrs in 1% water solution to fully activate the biological components of the productsand to allow the bacteria to get a quick start when introduced into the system. Soaking period of over 10-12 hrs are recommended.

In multiple – level applications, it is recommended that ENVIRO NZYME-G be applied at the lowest level first. Start at the bottom or end ofthe system from floor to floor or from section as appropriate. The reason for this is that where ENVIRO NZYME-G to be applied at the top orbeginning of the system, the waste material washed away might cause problems in the lower portion of the system unless this portion isalready cleared. During normal preventive maintenance, the point of application should be in an area which allows the product the max. timein the system.

HOW TO ADD THE ENVIRO NZYME-G INTO THE SYSTEM?

ENVIRO NZYME-G should be activated by mixing them in a bucket of warm water (not hot water) for few minutes. Pour the slurry into thesystem at a point where normal water flow will disperse it evenly. The best way is to pour it into the waste stream inside the plant, or justbefore it flows out into the waste stream inside the plant, or just before it flows out into the pond. If this is not possible, pour it directly intothe middle of the pond, or around the perimeter.

WHEN SHALL THE RESULTS BE ACHIEVED?

Bad odours and sludge accumulations take wonths and years to build up, and it will take some time to get rid of them. Begin the treatmentwith double the normal weekly dosage for a period of 3 to 4 weeks. After this start up period, one will begin to see significant improvements,and can cut back to normal weeklydose. Severe accumulation of sludge may take one or more years to get eliminated.

WHY TO HAVE A CONTINUOUS TREATMENT?

The bacteria in these products are very efficient at digesting waste-many time better then the natural bacteria. However, they are not as“strong” as the natural bacteria. If the treatment is stopped, the natural occurring bacteris are undesirable because they work slowly,produce odours and can be pathogenic.

WHICH OF THE DISINFECTANT CAN KILL BACTERIA?

Solutions: Do not chlorinate frequently. On application increase the dosage of ENVIRO NZYME-G.

OPERATIONAL PARAMETERS

ENVIRO NZYME-G contains a complex of bacteria and enzymes which perform in an adqueous solution within the following parameters:

pH

optimum 7.0 minimum 5.0 maximum 9.0

Dissolved Oxygen

optimum 2ppm minimum 1 ppm

(Should not be too acidic)

Carbon/Nitrogen

Ratio(C/N) Optimum 10:1 Maximum20:1

Temperature

optimum 30C minimum 10C maximum 40C

Negative effects on the biological growth rates are possible in the presence4 of varying concentrations of heavy metal in theadeeous solution (less than 20 ppm)

Residence time or the time biology needs to acclimatize to break down the organic waste is 5 days.

PRODUCT SPECIFICATION

BACTERIA COUNT

Aerobic BacteriaAnaerobic BacteriaFacultative BacteriaTotal plate CountE.ColiSalmonellaToxins

Min 1.5 billion colonies/gmMin 2.5 billion colonies/gmMin 2.0 billion colonies/gmMin 6.0 billion colonies/gmAbsent in 0.1 gmAbsent in 25 gmAbsent in 0.1 gm

APPEARANCE

Medium tan, free flowing granular powder with a yeasty odour.

STORAGE AND HANDLING

For a longer Shelf life keep in a dry storage area under 38C. Shelf life under these conditions estimated to be in excess of one year.

Temperature: More effective in our tropical lands

Avoid inhalation of dust and eye contact. Not for food use nor for contact with food preparatory surfaces o equipment.

Industrial Enzymes Share:We are leading manufacturers of Industrial Enzymes that comprise Batezyme, Enviro-Nzyme G,Cremo-Zyme, Earth-Nzyme, Tropizyme P, Agro Booster, Deter-Zyme X, Bio Feed, BakeryEnzymes and Textile Enzymes. With 25 years of rich industry experience backing our businessoperations, we are presently meeting the supply demands to Food, Agro, Detergent, Textile, Ice-cream and Effluent Treatment sector.

The details include:Chemical Compound

We provide various Chemical Compound for our clients.Also offering textile enzymes and bakery enzymes. Deter-Zyme X offered by us is alkaline protease enzyme that canbe used for broad spectrum of technical applications.

Alkaline and Acid Bate Deter Zyme X Textile EnzymesBetazyme

Agricultural Fertilizers

Send Enquiry

wide range of agricultural fertilizers that includes liquidbio-organic agricultural fertilizer, agro booster and naturalgrowth agricultural fertilizers. Also offering bio feed andbakery enzymes. Agro-Booster offered by us is liquid bio-organic fertilizer of plant origin (taken from 25 rare,medicinally important herbs).

Agro Booster Earth Nzyme

Poultry Feed EnzymePoultry Feed Enzyme that includes bio feed that includes

hygienic poultry feeds, poultry feed enzyme and nutritiouspoultry feed. Also offering textile enzymes and bakeryenzymes.

Bio Feed

Leather EnzymeCreating a niche of Leather Enzyme such as BIOBATE AC(Acid Bating Enzyme), Biobate AL (alkaline BatingEnzyme), Micrograin (Anti-wrinkle And Unhairing Enzymeand Soakzyme at its best, with utmost quality.

BIOBATE AC (Acid BatingEnzyme)

Biobate AL (alkaline BatingEnzyme)

Micrograin (Anti-wrinkleAnd Unhairing Enzyme

Soakzyme

Soy Protein Isolate

Soy Protein Isolate. Our product range also comprises ofChemical Compound, Agricultural Fertilizers and PoultryFeed Enzyme.

Soy Protein Isolate

Pharma Enzyme

Send Enquiry

Pharma Enzyme & PAPAIN. Our product range alsocomprises of Chemical Compound, Agricultural Fertilizersand Poultry Feed Enzyme.

PAPAIN

Brewing Enzymewide range of products which include Brewing Enzyme such

as THERMO-NZYME L and GLUCO-NZYME L.

THERMO-NZYME L GLUCO-NZYME L

Icecream Emulsifier StabilizerIcecream Emulsifier Stabilizer & Cremo Zyme. Our product

range also comprises of Chemical Compound, AgriculturalFertilizers and Poultry Feed Enzyme.

Cremo Zyme

Effluent TreatmentEffluent Treatment & Enviro Nzyme G. Our product range

also comprises of Chemical Compound, AgriculturalFertilizers and Poultry Feed Enzyme.

Enviro Nzyme G

Juice Enzyme

Juice Enzyme & Tropizyme P. Our product range alsocomprises of Chemical Compound, Agricultural Fertilizersand Poultry Feed Enzyme.

Tropizyme P

Bakery EnzymeSupplier & Distributor of Bakery Enzyme & BakeryEnzymes. Our product range also comprises of ChemicalCompound, Agricultural Fertilizers and Poultry FeedEnzyme.

EnzymeWebsite:www.fermentor.co.in

From Wikipedia, the free encyclopedia

"Biocatalyst" redirects here. For the use of natural catalysts in organic chemistry, see Biocatalysis.

Human glyoxalase I. Two zinc ions that are needed for the enzyme to catalyze its reaction are shown as purple spheres, and an enzyme inhibitor called S-hexylglutathione is shown as a space-filling model, filling the two active sites.

Enzymes ( /ˈɛnzaɪmz/) are biological molecules that catalyze (i.e., increase the rates of) chemical reactions.[1][2] In enzymatic

reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products.

Almost all chemical reactions in a biological cell need enzymes in order to occur at rates sufficient for life. Since enzymes are

selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell

determines which metabolic pathways occur in that cell.

Like all catalysts, enzymes work by lowering the activation energy (Ea‡) for a reaction, thus dramatically increasing the rate of the

reaction. As a result, products are formed faster and reactions reach their equilibrium state more rapidly. Most enzyme reaction

rates are millions of times faster than those of comparable un-catalyzed reactions. As with all catalysts, enzymes are not consumed

by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other

catalysts in that they are highly specific for their substrates. Enzymes are known to catalyze about 4,000 biochemical reactions.[3] A

few RNA molecules called ribozymes also catalyze reactions, with an important example being some parts of

the ribosome.[4][5] Synthetic molecules calledartificial enzymes also display enzyme-like catalysis.[6]

Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity; activators are

molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, pressure,

chemical environment (e.g., pH), and the concentration of substrate. Some enzymes are used commercially, for example, in the

synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in

biological washing powders break down protein or fat stains on clothes; enzymes in meat tenderizers break down proteins into

smaller molecules, making the meat easier to chew).

Contents

[hide]

1 Etymology and history

2 Structures and mechanisms

o 2.1 Specificity

2.1.1 "Lock and key" model

o 2.2 Mechanisms

2.2.1 Transition state stabilization

2.2.2 Dynamics and function

o 2.3 Allosteric modulation

3 Cofactors and coenzymes

o 3.1 Cofactors

o 3.2 Coenzymes

4 Thermodynamics

5 Kinetics

6 Inhibition

7 Biological function

8 Control of activity

9 Involvement in disease

10 Naming conventions

11 Industrial applications

12 See also

13 References

14 Further reading

15 External links

[edit]Etymology and history

Eduard Buchner

As early as the late 17th and early 18th centuries, the digestion of meat by stomach secretions[7] and the conversion

of starch to sugars by plant extracts andsaliva were known. However, the mechanism by which this occurred had not been

identified.[8]

In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this

fermentation was catalyzed by a vital force contained within the yeast cells called "ferments", which were thought to function only

within living organisms. He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not

with the death or putrefaction of the cells."[9]

In 1877, German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme, which comes from Greek ενζυμον, "in

leaven", to describe this process.[10] The word enzyme was used later to refer to nonliving substances such as pepsin, and the

word ferment was used to refer to chemical activity produced by living organisms.

In 1897, Eduard Buchner submitted his first paper on the ability of yeast extracts that lacked any living yeast cells to ferment sugar.

In a series of experiments at the University of Berlin, he found that the sugar was fermented even when there were no living yeast

cells in the mixture.[11] He named the enzyme that brought about the fermentation of sucrose "zymase".[12] In 1907, he received

the Nobel Prize in Chemistry "for his biochemical research and his discovery of cell-free fermentation". Following Buchner's

example, enzymes are usually named according to the reaction they carry out. Typically, to generate the name of an enzyme, the

suffix -ase is added to the name of its substrate (e.g., lactase is the enzyme that cleaves lactose) or the type of reaction (e.g., DNA

polymerase forms DNA polymers).[13]

Having shown that enzymes could function outside a living cell, the next step was to determine their biochemical nature. Many early

workers noted that enzymatic activity was associated with proteins, but several scientists (such as Nobel laureate Richard

Willstätter) argued that proteins were merely carriers for the true enzymes and that proteinsper se were incapable of

catalysis.[14] However, in 1926, James B. Sumner showed that the enzyme urease was a pure protein and crystallized it; Sumner did

likewise for the enzyme catalase in 1937. The conclusion that pure proteins can be enzymes was definitively proved

by Northrop and Stanley, who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin. These three scientists

were awarded the 1946 Nobel Prize in Chemistry.[15]

This discovery that enzymes could be crystallized eventually allowed their structures to be solved by x-ray crystallography. This was

first done for lysozyme, an enzyme found in tears, saliva andegg whites that digests the coating of some bacteria; the structure was

solved by a group led by David Chilton Phillips and published in 1965.[16] This high-resolution structure of lysozyme marked the

beginning of the field of structural biology and the effort to understand how enzymes work at an atomic level of detail.

[edit]Structures and mechanisms

See also: Enzyme catalysis

Ribbon diagram showing human carbonic anhydrase II. The grey sphere is the zinc cofactor in the active site. Diagram drawn from PDB 1MOO.

Enzymes are in general globular proteins and range from just 62 amino acid residues in size, for the monomer of 4-oxalocrotonate

tautomerase,[17] to over 2,500 residues in the animal fatty acid synthase.[18] A small number of RNA-based biological catalysts exist,

with the most common being the ribosome; these are referred to as either RNA-enzymes or ribozymes. The activities of enzymes

are determined by their three-dimensional structure.[19] However, although structure does determine function, predicting a novel

enzyme's activity just from its structure is a very difficult problem that has not yet been solved.[20]

Most enzymes are much larger than the substrates they act on, and only a small portion of the enzyme (around 2–4 amino acids) is

directly involved in catalysis.[21] The region that contains these catalytic residues, binds the substrate, and then carries out the

reaction is known as the active site. Enzymes can also contain sites that bind cofactors, which are needed for catalysis. Some

enzymes also have binding sites for small molecules, which are often direct or indirect products or substrates of the reaction

catalyzed. This binding can serve to increase or decrease the enzyme's activity, providing a means for feedback regulation.

Like all proteins, enzymes are long, linear chains of amino acids that fold to produce a three-dimensional product. Each unique

amino acid sequence produces a specific structure, which has unique properties. Individual protein chains may sometimes group

together to form aprotein complex. Most enzymes can be denatured—that is, unfolded and inactivated—by heating or chemical

denaturants, which disrupt the three-dimensional structure of the protein. Depending on the enzyme, denaturation may be reversible

or irreversible.

Structures of enzymes with substrates or substrate analogs during a reaction may be obtained using Time resolved

crystallographymethods.

[edit]Specificity

Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions.

Complementary shape, charge and hydrophilic/hydrophobiccharacteristics of enzymes and substrates are responsible for this

specificity. Enzymes can also show impressive levels of stereospecificity, regioselectivity and chemoselectivity.[22]

Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome.

These enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA polymerase catalyzes a reaction in a first step

and then checks that the product is correct in a second step.[23] This two-step process results in average error rates of less than 1

error in 100 million reactions in high-fidelity mammalian polymerases.[24] Similar proofreading mechanisms are also found in RNA

polymerase,[25] aminoacyl tRNA synthetases[26] andribosomes.[27]

Some enzymes that produce secondary metabolites are described as promiscuous, as they can act on a relatively broad range of

different substrates. It has been suggested that this broad substrate specificity is important for the evolution of new biosynthetic

pathways.[28]

[edit]"Lock and key" model

Enzymes are very specific, and it was suggested by the Nobel laureate organic chemist Emil Fischer in 1894 that this was because

both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another.[29] This is

often referred to as "the lock and key" model. However, while this model explains enzyme specificity, it fails to explain the

stabilization of the transition state that enzymes achieve.

Diagrams to show the induced fit hypothesis of enzyme action

In 1958, Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the

active site is continuously reshaped by interactions with the substrate as the substrate interacts with the enzyme.[30] As a result, the

substrate does not simply bind to a rigid active site; the amino acid side-chains that make up the active site are molded into the

precise positions that enable the enzyme to perform its catalytic function. In some cases, such as glycosidases, the substrate

molecule also changes shape slightly as it enters the active site.[31] The active site continues to change until the substrate is

completely bound, at which point the final shape and charge is determined.[32] Induced fit may enhance the fidelity of molecular

recognition in the presence of competition and noise via the conformational proofreading mechanism.[33]

[edit]Mechanisms

Enzymes can act in several ways, all of which lower ΔG‡ (Gibbs energy):[34]

Lowering the activation energy by creating an environment in which the transition state is stabilized (e.g. straining the

shape of a substrate—by binding the transition-state conformation of the substrate/product molecules, the enzyme distorts the

bound substrate(s) into their transition state form, thereby reducing the amount of energy required to complete the transition).

Lowering the energy of the transition state, but without distorting the substrate, by creating an environment with the

opposite charge distribution to that of the transition state.

Providing an alternative pathway. For example, temporarily reacting with the substrate to form an intermediate ES

complex, which would be impossible in the absence of the enzyme.

Reducing the reaction entropy change by bringing substrates together in the correct orientation to react. Considering

ΔH‡ alone overlooks this effect.

Increases in temperatures speed up reactions. Thus, temperature increases help the enzyme function and develop the

end product even faster. However, if heated too much, the enzyme’s shape deteriorates and the enzyme becomes denatured.

Some enzymes like thermolabile enzymes work best at low temperatures.

It is interesting that this entropic effect involves destabilization of the ground state,[35] and its contribution to catalysis is relatively

small.[36]

[edit]Transition state stabilization

The understanding of the origin of the reduction of ΔG‡ requires one to find out how the enzymes can stabilize its transition state

more than the transition state of the uncatalyzed reaction. It seems that the most effective way for reaching large stabilization is the

use of electrostatic effects, in particular, when having a relatively fixed polar environment that is oriented toward the charge

distribution of the transition state.[37] Such an environment does not exist in the uncatalyzed reaction in water.

[edit]Dynamics and function

See also: Protein dynamics

The internal dynamics of enzymes has been suggested to be linked with their mechanism of catalysis.[38][39][40] Internal dynamics are

the movement of parts of the enzyme's structure, such as individual amino acid residues, a group of amino acids, or even an

entire protein domain. These movements occur at various time-scales ranging from femtoseconds to seconds. Networks of protein

residues throughout an enzyme's structure can contribute to catalysis through dynamic motions.[41][42][43][44] This is simply seen in

the kinetic scheme of the combined process, enzymatic activity and dynamics; this scheme can have several

independent Michaelis-Menten-like reaction pathways that are connected through fluctuation rates.[45][46][47]

Protein motions are vital to many enzymes, but whether small and fast vibrations, or larger and slower conformational movements

are more important depends on the type of reaction involved. However, although these movements are important in binding and

releasing substrates and products, it is not clear if protein movements help to accelerate the chemical steps in enzymatic

reactions.[48] These new insights also have implications in understanding allosteric effects and developing new medicines.

[edit]Allosteric modulation

Allosteric transition of an enzyme between R and T states, stabilized by an agonist, an inhibitor and a substrate (the MWC model)

Main article: Allosteric regulation

Allosteric sites are sites on the enzyme that bind to molecules in the cellular environment. The sites form weak, noncovalent bonds

with these molecules, causing a change in the conformation of the enzyme. This change in conformation translates to the active

site, which then affects the reaction rate of the enzyme.[49] Allosteric interactions can both inhibit and activate enzymes and are a

common way that enzymes are controlled in the body.[50]

[edit]Cofactors and coenzymes

Main articles: Cofactor (biochemistry) and Coenzyme

[edit]Cofactors

Some enzymes do not need any additional components to show full activity. However, others require non-protein molecules called

cofactors to be bound for activity.[51] Cofactors can be either inorganic (e.g., metal ions and iron-sulfur clusters) or organic

compounds (e.g., flavin and heme). Organic cofactors can be either prosthetic groups, which are tightly bound to an enzyme,

or coenzymes, which are released from the enzyme's active site during the reaction. Coenzymes

include NADH, NADPH and adenosine triphosphate. These molecules transfer chemical groups between enzymes.[52]

An example of an enzyme that contains a cofactor is carbonic anhydrase, and is shown in the ribbon diagram above with a zinc

cofactor bound as part of its active site.[53] These tightly bound molecules are usually found in the active site and are involved in

catalysis. For example, flavin and heme cofactors are often involved in redox reactions.

Enzymes that require a cofactor but do not have one bound are called apoenzymes or apoproteins. An apoenzyme together with its

cofactor(s) is called a holoenzyme (this is the active form). Most cofactors are not covalently attached to an enzyme, but are very

tightly bound. However, organic prosthetic groups can be covalently bound (e.g., biotin in the enzyme pyruvate carboxylase). The

term "holoenzyme" can also be applied to enzymes that contain multiple protein subunits, such as the DNA polymerases; here the

holoenzyme is the complete complex containing all the subunits needed for activity.

[edit]Coenzymes

Space-filling model of the coenzyme NADH

Coenzymes are small organic molecules that can be loosely or tightly bound to an enzyme. Tightly bound coenzymes can be called

allosteric groups. Coenzymes transport chemical groups from one enzyme to another.[54] Some of these chemicals such

as riboflavin, thiamine and folic acid are vitamins (compounds that cannot be synthesized by the body and must be acquired from

the diet). The chemical groups carried include the hydride ion (H-) carried by NAD or NADP+, the phosphate group carried

by adenosine triphosphate, the acetyl group carried by coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the

methyl group carried by S-adenosylmethionine.

Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special

class of substrates, or second substrates, which are common to many different enzymes. For example, about 700 enzymes are

known to use the coenzyme NADH.[55]

Coenzymes are usually continuously regenerated and their concentrations maintained at a steady level inside the cell: for example,

NADPH is regenerated through the pentose phosphate pathway and S-adenosylmethionine by methionine adenosyltransferase.

This continuous regeneration means that even small amounts of coenzymes are used very intensively. For example, the human

body turns over its own weight in ATP each day.[56]

[edit]Thermodynamics

The energies of the stages of a chemical reaction. Substrates need a lot of potential energy to reach a transition state, which then decays into products. The enzyme stabilizes the transition state, reducing the energy needed to form products.

Main articles: Activation energy, Thermodynamic equilibrium, and Chemical equilibrium

As all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction. Usually, in the presence of an enzyme,

the reaction runs in the same direction as it would without the enzyme, just more quickly. However, in the absence of the enzyme,

other possible uncatalyzed, "spontaneous" reactions might lead to different products, because in those conditions this different

product is formed faster.

Furthermore, enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to "drive" a

thermodynamically unfavorable one. For example, the hydrolysis of ATP is often used to drive other chemical reactions.[57]

Enzymes catalyze the forward and backward reactions equally. They do not alter the equilibrium itself, but only the speed at which it

is reached. For example, carbonic anhydrase catalyzes its reaction in either direction depending on the concentration of its

reactants.

(in tissues; high CO2 concentration)

(in lungs; low CO2 concentration)

Nevertheless, if the equilibrium is greatly displaced in one direction, that is, in a very exergonic reaction, the reaction is in

effect irreversible. Under these conditions, the enzyme will, in fact, catalyze the reaction only in the thermodynamically

allowed direction.

[edit]Kinetics

Main article: Enzyme kinetics

Mechanism for a single substrate enzyme catalyzed reaction. The enzyme (E) binds a substrate (S) and produces a product (P).

Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in

kinetic analyses are commonly obtained from enzyme assays, where since the 90s, the dynamics of many enzymes are

studied on the level of individual molecules.

In 1902 Victor Henri proposed a quantitative theory of enzyme kinetics,[58] but his experimental data were not useful

because the significance of the hydrogen ion concentration was not yet appreciated. After Peter Lauritz Sørensen had

defined the logarithmic pH-scale and introduced the concept of buffering in 1909[59] the German chemist Leonor

Michaelis and his Canadian postdoc Maud Leonora Menten repeated Henri's experiments and confirmed his equation,

which is referred to as Henri-Michaelis-Menten kinetics (termed alsoMichaelis-Menten kinetics).[60] Their work was further

developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic equations that are still widely considered today a

starting point in solving enzymatic activity.[61]

The major contribution of Henri was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly

to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis complex. The enzyme

then catalyzes the chemical step in the reaction and releases the product. Note that the simple Michaelis Menten

mechanism for the enzymatic activity is considered today a basic idea, where many examples show that the enzymatic

activity involves structural dynamics. This is incorporated in the enzymatic mechanism while introducing several Michaelis

Menten pathways that are connected with fluctuating rates.[45][46][47] Nevertheless, there is a mathematical relation

connecting the behavior obtained from the basic Michaelis Menten mechanism (that was indeed proved correct in many

experiments) with the generalized Michaelis Menten mechanisms involving dynamics and activity; [62] this means that the

measured activity of enzymes on the level of many enzymes may be explained with the simple Michaelis-Menten

equation, yet, the actual activity of enzymes is richer and involves structural dynamics.

Saturation curve for an enzyme reaction showing the relation between the substrate concentration (S) and rate (v)

Enzymes can catalyze up to several million reactions per second. For example, the uncatalyzed decarboxylation

of orotidine 5'-monophosphate has a half life of 78 million years. However, when the enzyme orotidine 5'-phosphate

decarboxylase is added, the same process takes just 25 milliseconds.[63] Enzyme rates depend on solution conditions

and substrate concentration. Conditions that denature the protein abolish enzyme activity, such as high temperatures,

extremes of pH or high salt concentrations, while raising substrate concentration tends to increase activity when [S] is

low. To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate

of product formation is seen. This is shown in the saturation curve on the right. Saturation happens because, as substrate

concentration increases, more and more of the free enzyme is converted into the substrate-bound ES form. At the

maximum reaction rate (Vmax) of the enzyme, all the enzyme active sites are bound to substrate, and the amount of ES

complex is the same as the total amount of enzyme. However, Vmax is only one kinetic constant of enzymes. The amount

of substrate needed to achieve a given rate of reaction is also important. This is given by the Michaelis-Menten

constant (Km), which is the substrate concentration required for an enzyme to reach one-half its maximum reaction rate.

Each enzyme has a characteristic Km for a given substrate, and this can show how tight the binding of the substrate is to

the enzyme. Another useful constant is kcat, which is the number of substrate molecules handled by one active site per

second.

The efficiency of an enzyme can be expressed in terms of kcat/Km. This is also called the specificity constant and

incorporates the rate constants for all steps in the reaction. Because the specificity constant reflects both affinity and

catalytic ability, it is useful for comparing different enzymes against each other, or the same enzyme with different

substrates. The theoretical maximum for the specificity constant is called the diffusion limit and is about 108 to

109 (M−1s−1). At this point every collision of the enzyme with its substrate will result in catalysis, and the rate of product

formation is not limited by the reaction rate but by the diffusion rate. Enzymes with this property are called catalytically

perfect or kinetically perfect. Example of such enzymes are triose-phosphate isomerase, carbonic

anhydrase, acetylcholinesterase, catalase, fumarase, β-lactamase, and superoxide dismutase.

Michaelis-Menten kinetics relies on the law of mass action, which is derived from the assumptions of free diffusion and

thermodynamically driven random collision. However, many biochemical or cellular processes deviate significantly from

these conditions, because of macromolecular crowding, phase-separation of the enzyme/substrate/product, or one or

two-dimensional molecular movement.[64] In these situations, a fractal Michaelis-Menten kinetics may be

applied.[65][66][67][68]

Some enzymes operate with kinetics, which are faster than diffusion rates, which would seem to be impossible. Several

mechanisms have been invoked to explain this phenomenon. Some proteins are believed to accelerate catalysis by

drawing their substrate in and pre-orienting them by using dipolar electric fields. Other models invoke a quantum-

mechanical tunneling explanation, whereby a proton or an electron can tunnel through activation barriers, although for

proton tunneling this model remains somewhat controversial.[69][70] Quantum tunneling for protons has been observed

in tryptamine.[71] This suggests that enzyme catalysis may be more accurately characterized as "through the barrier"

rather than the traditional model, which requires substrates to go "over" a lowered energy barrier.

[edit]Inhibition

Competitive inhibitors bind reversibly to the enzyme, preventing the binding of substrate. On the other hand, binding of substrate prevents binding of the inhibitor. Substrate and inhibitor compete for the enzyme.

Types of inhibition. This classification was introduced by W.W. Cleland.[72]

Main article: Enzyme inhibitor

Enzyme reaction rates can be decreased by various types of enzyme inhibitors.

Competitive inhibition

In competitive inhibition, the inhibitor and substrate compete for the enzyme (i.e., they can not bind at the same

time).[73] Often competitive inhibitors strongly resemble the real substrate of the enzyme. For example, methotrexate is a

competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction

of dihydrofolate totetrahydrofolate. The similarity between the structures of folic acid and this drug are shown in the figure

to the rightbottom. In some cases, the inhibitor can bind to a site other than the binding-site of the usual substrate and

exert anallosteric effect to change the shape of the usual binding-site. For example, strychnine acts as an allosteric

inhibitor of the glycine receptor in the mammalian spinal cord and brain stem. Glycine is a major post-synaptic inhibitory

neurotransmitter with a specific receptor site. Strychnine binds to an alternate site that reduces the affinity of the glycine

receptor for glycine, resulting in convulsions due to lessened inhibition by the glycine.[74] In competitive inhibition the

maximal rate of the reaction is not changed, but higher substrate concentrations are required to reach a given maximum

rate, increasing the apparent Km.

Uncompetitive inhibition

In uncompetitive inhibition, the inhibitor cannot bind to the free enzyme, only to the ES-complex. The EIS-complex thus

formed is enzymatically inactive. This type of inhibition is rare, but may occur in multimeric enzymes.

Non-competitive inhibition

Non-competitive inhibitors can bind to the enzyme at the binding site at the same time as the substrate,but not to the

active site. Both the EI and EIS complexes are enzymatically inactive. Because the inhibitor can not be driven from the

enzyme by higher substrate concentration (in contrast to competitive inhibition), the apparent Vmax changes. But because

the substrate can still bind to the enzyme, the Km stays the same.

Mixed inhibition

This type of inhibition resembles the non-competitive, except that the EIS-complex has residual enzymatic activity.This

type of inhibitor does not follow Michaelis-Menten equation.

In many organisms, inhibitors may act as part of a feedback mechanism. If an enzyme produces too much of one

substance in the organism, that substance may act as an inhibitor for the enzyme at the beginning of the pathway that

produces it, causing production of the substance to slow down or stop when there is sufficient amount. This is a form

ofnegative feedback. Enzymes that are subject to this form of regulation are often multimeric and have allosteric binding

sites for regulatory substances. Their substrate/velocity plots are not hyperbolar, but sigmoidal (S-shaped).

The coenzyme folic acid (left) and the anti-cancer drug methotrexate (right) are very similar in structure. As a result, methotrexate is a competitive inhibitor of many enzymes that use folates.

Irreversible inhibitors react with the enzyme and form a covalent adduct with the protein. The inactivation is irreversible.

These compounds include eflornithine a drug used to treat the parasitic disease sleeping

sickness.[75] Penicillin andAspirin also act in this manner. With these drugs, the compound is bound in the active site and

the enzyme then converts the inhibitor into an activated form that reacts irreversibly with one or more amino acid

residues.

Uses of inhibitors

Since inhibitors modulate the function of enzymes they are often used as drugs. A common example of an inhibitor that is

used as a drug is aspirin, which inhibits the COX-1 and COX-2 enzymes that produce

the inflammation messengerprostaglandin, thus suppressing pain and inflammation. However, other enzyme inhibitors

are poisons. For example, the poison cyanide is an irreversible enzyme inhibitor that combines with the copper and iron

in the active site of the enzymecytochrome c oxidase and blocks cellular respiration.[76]

[edit]Biological function

Enzymes serve a wide variety of functions inside living organisms. They are indispensable for signal transduction and cell

regulation, often via kinases and phosphatases.[77] They also generate movement, with myosin hydrolyzing ATP to

generate muscle contraction and also moving cargo around the cell as part of the cytoskeleton.[78] Other ATPases in the

cell membrane are ion pumps involved in active transport. Enzymes are also involved in more exotic functions, such

asluciferase generating light in fireflies.[79] Viruses can also contain enzymes for infecting cells, such as the HIV

integraseand reverse transcriptase, or for viral release from cells, like the influenza virus neuraminidase.

An important function of enzymes is in the digestive systems of animals. Enzymes such as amylases and proteasesbreak

down large molecules (starch or proteins, respectively) into smaller ones, so they can be absorbed by the intestines.

Starch molecules, for example, are too large to be absorbed from the intestine, but enzymes hydrolyze the starch chains

into smaller molecules such as maltose and eventually glucose, which can then be absorbed. Different enzymes digest

different food substances. In ruminants, which have herbivorous diets, microorganisms in the gut produce another

enzyme, cellulase, to break down the cellulose cell walls of plant fiber.[80]

Glycolytic enzymes and their functions in the metabolic pathway of glycolysis

Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic pathway, one

enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to

another enzyme. Sometimes more than one enzyme can catalyze the same reaction in parallel; this can allow more

complex regulation: with, for example, a low constant activity provided by one enzyme but an inducible high activity from

a second enzyme.

Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through

the same steps nor be fast enough to serve the needs of the cell. Indeed, a metabolic pathway such as glycolysis could

not exist independently of enzymes. Glucose, for example, can react directly with ATP to become phosphorylated at one

or more of its carbons. In the absence of enzymes, this occurs so slowly as to be insignificant. However, if hexokinase is

added, these slow reactions continue to take place except that phosphorylation at carbon 6 occurs so rapidly that, if the

mixture is tested a short time later, glucose-6-phosphate is found to be the only significant product. As a consequence,

the network of metabolic pathways within each cell depends on the set of functional enzymes that are present.

[edit]Control of activity

There are five main ways that enzyme activity is controlled in the cell.

1. Enzyme production (transcription and translation of enzyme genes) can be enhanced or diminished by a cell

in response to changes in the cell's environment. This form of gene regulationis called enzyme induction and

inhibition. For example, bacteria may become resistant to antibiotics such as penicillin because enzymes

called beta-lactamases are induced that hydrolyze the crucial beta-lactam ring within the penicillin molecule.

Another example are enzymes in the liver called cytochrome P450 oxidases, which are important in drug

metabolism. Induction or inhibition of these enzymes can cause drug interactions.

2. Enzymes can be compartmentalized, with different metabolic pathways occurring in different cellular

compartments. For example, fatty acids are synthesized by one set of enzymes in the cytosol, endoplasmic

reticulum and the Golgi apparatus and used by a different set of enzymes as a source of energy in

the mitochondrion, through β-oxidation.[81]

3. Enzymes can be regulated by inhibitors and activators. For example, the end product(s) of a metabolic

pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step,

called committed step), thus regulating the amount of end product made by the pathways. Such a regulatory

mechanism is called a negative feedback mechanism, because the amount of the end product produced is

regulated by its own concentration. Negative feedback mechanism can effectively adjust the rate of synthesis

of intermediate metabolites according to the demands of the cells. This helps allocate materials and energy

economically, and prevents the manufacture of excess end products. The control of enzymatic action helps to

maintain a stable internal environment in living organisms.

4. Enzymes can be regulated through post-translational modification. This can

include phosphorylation, myristoylation and glycosylation. For example, in the response to insulin,

thephosphorylation of multiple enzymes, including glycogen synthase, helps control the synthesis or

degradation of glycogen and allows the cell to respond to changes in blood sugar.[82]Another example of post-

translational modification is the cleavage of the polypeptide chain. Chymotrypsin, a digestive protease, is

produced in inactive form as chymotrypsinogen in thepancreas and transported in this form to

the stomach where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it

enters the gut. This type of inactive precursor to an enzyme is known as a zymogen.

5. Some enzymes may become activated when localized to a different environment (e.g., from a reducing

(cytoplasm) to an oxidizing (periplasm) environment, high pH to low pH, etc.). For example, hemagglutinin in

the influenza virus is activated by a conformational change caused by the acidic conditions, these occur when

it is taken up inside its host cell and enters the lysosome.[83]

[edit]Involvement in disease

Phenylalanine hydroxylase. Created from PDB 1KW0

Since the tight control of enzyme activity is essential for homeostasis, any malfunction (mutation, overproduction,

underproduction or deletion) of a single critical enzyme can lead to a genetic disease. The importance of enzymes is

shown by the fact that a lethal illness can be caused by the malfunction of just one type of enzyme out of the thousands

of types present in our bodies.

One example is the most common type of phenylketonuria. A mutation of a single amino acid in the

enzyme phenylalanine hydroxylase, which catalyzes the first step in the degradation of phenylalanine, results in build-up

of phenylalanine and related products. This can lead to mental retardation if the disease is untreated.[84]

Another example of enzyme deficiency is pseudocholinesterase, in which there is slow metabolic degradation of

exogenous choline.[citation needed]

Another example is when germline mutations in genes coding for DNA repair enzymes cause hereditary cancer

syndromes such as xeroderma pigmentosum. Defects in these enzymes cause cancer since the body is less able to

repair mutations in the genome. This causes a slow accumulation of mutations and results in the development of many

types of cancer in the sufferer.

Oral administration of enzymes can be used to treat several diseases (e.g. pancreatic insufficiency and lactose

intolerance). Since enzymes are proteins themselves they are potentially subject to inactivation and digestion in the

gastrointestinal environment. Therefore a non-invasive imaging assay was developed to monitor gastrointestinal activity

of exogenous enzymes (prolyl endopeptidase as potential adjuvant therapy for celiac disease) in vivo.[85]

[edit]Naming conventions

An enzyme's name is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase.

Examples are lactase, alcohol dehydrogenase and DNA polymerase. This may result in different enzymes,

called isozymes, with the same function having the same basic name. Isoenzymes have a different amino acid sequence

and might be distinguished by their optimal pH, kinetic properties or immunologically. Isoenzyme and isozyme are

homologous proteins. Furthermore, the normal physiological reaction an enzyme catalyzes may not be the same as

under artificial conditions. This can result in the same enzyme being identified with two different names. For

example, glucose isomerase, which is used industrially to convert glucose into the sweetener fructose, is a xylose

isomerase in vivo (within the body).

The International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes, the EC

numbers; each enzyme is described by a sequence of four numbers preceded by "EC". The first number broadly

classifies the enzyme based on its mechanism.

The top-level classification is[86]

EC 1 Oxidoreductases: catalyze oxidation/reduction reactions

EC 2 Transferases: transfer a functional group (e.g. a methyl or phosphate group)

EC 3 Hydrolases: catalyze the hydrolysis of various bonds

EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation

EC 5 Isomerases: catalyze isomerization changes within a single molecule

EC 6 Ligases: join two molecules with covalent bonds.

According to the naming conventions, enzymes are generally classified into six main family classes and many sub-family

classes. Some web-servers, e.g., EzyPred [87] and bioinformatics tools have been developed to predict which main family

class [88] and sub-family class [89] [90] an enzyme molecule belongs to according to its sequence information alone via

the pseudo amino acid composition.

[edit]Industrial applications

Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are

required. However, enzymes in general are limited in the number of reactions they have evolved to catalyze and also by

their lack of stability in organic solvents and at high temperatures. As a consequence, protein engineering is an active

area of research and involves attempts to create new enzymes with novel properties, either through rational design or in

vitro evolution.[91][92] These efforts have begun to be successful, and a few enzymes have now been designed "from

scratch" to catalyze reactions that do not occur in nature.[93]

Application Enzymes used Uses

Food processing

Amylases catalyze the release of

simple sugars from starch.

Amylases from fungi and plants

Production of sugars from starch,such as in making high-fructosecorn syrup.[94] In baking, catalyzebreakdown of starch inthe flour to sugar. Yeastfermentation of sugar producesthe carbon dioxide that raises thedough.

ProteasesBiscuit manufacturers use them tolower the protein level of flour.

Baby foods Trypsin To predigest baby foods

Brewing industry

Germinating barley used for

malt

Enzymes from barley are releasedduring the mashing stage of beerproduction.

They degrade starch and proteinsto produce simple sugar, aminoacids and peptides that are usedby yeast for fermentation.

Industrially produced barley enzymesWidely used in the brewingprocess to substitute for thenatural enzymes found in barley.

Amylase, glucanases, proteasesSplit polysaccharides andproteins in the malt.

Betaglucanases and arabinoxylanasesImprove the wort and beerfiltration characteristics.

Amyloglucosidase and pullulanasesLow-calorie beer and adjustmentof fermentability.

ProteasesRemove cloudiness producedduring storage of beers.

Acetolactatedecarboxylase (ALDC)Increases fermentation efficiencybyreducing diacetyl formation.[95]

Fruit juices Cellulases, pectinases Clarify fruit juices.

Dairy industry

Roquefort cheese

Rennin, derived from the stomachs ofyoung ruminant animals (like calvesand lambs)

Manufacture of cheese, usedto hydrolyze protein

Microbially produced enzymeNow finding increasing use in thedairy industry

Lipases

Is implemented during theproduction of Roquefort cheese toenhance the ripening of the blue-mold cheese.

LactasesBreakdown lactose to glucose andgalactose.

Meat tenderizers Papain To soften meat for cooking

Starch industry

Glucose Fructose

Amylases, amyloglucosideases andglucoamylases

Converts starch into glucose andvarious syrups.

Glucose isomerase

Converts glucose into fructose inproduction of high-fructosesyrups from starchy materials.These syrups have enhancedsweetening properties andlower calorific values thansucrose for the same level ofsweetness.

Paper industry

A paper mill in South Carolina

Amylases, Xylanases, Cellulases andligninases

Degrade starch to lower viscosity,aiding sizing and coating paper.Xylanases reduce bleach requiredfor decolorizing; cellulasessmooth fibers, enhance waterdrainage, and promote inkremoval; lipases reduce pitch andlignin-degrading enzymesremove lignin to soften paper.

Biofuel industry

Cellulose in 3D

CellulasesUsed to break down cellulose intosugars that can be fermented(seecellulosic ethanol)

Ligninases Use of lignin waste

Biological detergent

Primarily proteases, produced inan extracellular form from bacteria

Used for presoak conditions anddirect liquid applications helpingwith removal of protein stainsfrom clothes

AmylasesDetergents for machine dishwashing to remove resistantstarch residues

LipasesUsed to assist in the removal offatty and oily stains

CellulasesUsed in biological fabricconditioners

Contact lens cleaners ProteasesTo remove proteins on contactlens to prevent infections

Rubber industry CatalaseTogenerate oxygen from peroxide toconvert latex into foam rubber

Photographic industry Protease (ficin)Dissolve gelatin off scrap film,allowing recovery ofits silver content.

Molecular biology

Part of the DNA double helix

Restriction enzymes, DNAligase and polymerases

Used to manipulate DNAin genetic engineering, importantinpharmacology, agriculture andmedicine. Essential for restrictiondigestion and the polymerasechain reaction. Molecular biologyis also important in forensicscience.

[edit]See also

Food portal

List of enzymes

Enzyme product

Product (biology)From Wikipedia, the free encyclopedia

This article does not cite any references or sources. Please help improve this article by adding citations to reliablesources. Unsourced material may be challenged and removed. (December 2009)

In biochemistry, a product is something "manufactured" by an enzyme from its substrate. An example of this would

be;the products of lactase are galactose and glucose, which are produced from the substrate lactose.

Reaction using lactase

[edit]

Enzyme substrate

Enzyme substrate (biology)From Wikipedia, the free encyclopedia

(Redirected from Enzyme substrate)

For other uses, see Substrate (disambiguation).

In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical

reactions involving the substrate(s). In the case of a single substrate, the substrate binds with the

enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or

more products, which are then released from the active site. The active site is now free to accept another

substrate molecule. In the case of more than one substrate, these may bind in a particular order to the active

site, before reacting together to produce products.

For example, curd formation (rennet coagulation) is a reaction that occurs upon adding the enzyme rennin to

milk. In this reaction, the substrate is a milk protein (e.g., casein) and the enzyme is rennin. The products are

two polypeptides that have been formed by the cleavage of the larger peptide substrate. Another example is

the chemical decomposition of hydrogen peroxide carried out by the enzyme catalase. As enzymes

are catalysts, they are not changed by the reactions they carry out. The substrate(s), however, is/are converted

to product(s). Here, hydrogen peroxide is converted to water and oxygen gas.

E + S ⇌ ES → E+ P

where E = enzyme, S = substrate(s), P = product(s). While the first (binding) and third (unbinding) steps

are, in general, reversible, the middle step may be irreversible (as in the rennin and catalase reactions just

mentioned) or reversible (e.g., many reactions in the glycolysis metabolic pathway).

By increasing the substrate concentration, the rate of reaction will increase due to the likelihood that the

number of enzyme-substrate complexes will increase; this occurs until the enzymeconcentration becomes

the limiting factor.

It is important to note that the substrates that a given amino acid in vitro may not necessarily reflect the

physiological, endogenous substrates of the enzyme in vivo. That is to say that enzymes do not

necessarily perform all the reactions in the body that may be possible in the laboratory. For example,

while fatty acid amide hydrolase (FAAH) can hydrolyze the endocannabinoids 2-arachidonoylglycerol (2-

AG) and anandamide at comparable rates in vitro, genetic or pharmacological disruption of FAAH elevates

anandamide but not 2-AG, suggesting that 2-AG is not an endogenous, in vivo substrate for FAAH.[1] In

another example, the N-acyl taurines (NATs) are observed to increase dramatically in FAAH-disrupted

animals, but are actually poor in vitro FAAH substrates.[2]

Enzyme catalysis

Enzyme catalysisFrom Wikipedia, the free encyclopedia

Enzyme catalysis is the catalysis of chemical reactions by specialized proteins known as enzymes. Catalysis

of biochemical reactions in the cell is vital due to the very low reaction rates of the uncatalysed reactions.

The mechanism of enzyme catalysis is similar in principle to other types of chemical catalysis. By providing an alternative

reaction route and by stabilizing intermediates the enzyme reduces the energy required to reach the highest

energy transition state of the reaction. The reduction of activation energy (Ea) increases the number of reactant molecules

with enough energy to reach the activation energy and form the product.

Contents

[hide]

1 Induced fit

o 1.1 Catalysis by induced fit

2 Mechanisms of transition state stabilization

o 2.1 Catalysis by bond strain

o 2.2 Catalysis by proximity and orientation

o 2.3 Catalysis involving proton donors or acceptors (Acid/Base Catalysis)

o 2.4 Electrostatic catalysis

o 2.5 Covalent catalysis

o 2.6 Quantum tunneling

3 Examples of catalytic mechanisms

o 3.1 Triose phosphate isomerase

o 3.2 Trypsin

o 3.3 Aldolase

4 See also

5 References

6 Further reading

[edit]Induced fit

Diagrams to show the induced fit hypothesis of enzyme action.

The favored model for the enzyme-substrate interaction is the induced fit model.[1] This model proposes that the initial

interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly

induce conformational changes in the enzyme that strengthen binding.

[edit]Catalysis by induced fit

The different mechanisms of substrate binding

The advantages of the induced fit mechanism arise due to the stabilizing effect of strong enzyme binding. There are two

different mechanisms of substrate binding: uniform binding, which has strong substrate binding, and differential binding,

which has strong transition state binding. The stabilizing effect of uniform binding increases both substrate and transition

state binding affinity, while differential binding increases only transition state binding affinity. Both are used by enzymes

and have been evolutionarily chosen to minimize the Ea of the reaction. Enzymes which are saturated, that is, have a high

affinity substrate binding, require differential binding to reduce the Ea, whereas small substrate unbound enzymes may use

either differential or uniform binding.

These effects have led to most proteins using the differential binding mechanism to reduce the Ea, so most proteins have

high affinity of the enzyme to the transition state. Differential binding is carried out by the induced fit mechanism - the

substrate first binds weakly, then the enzyme changes conformation increasing the affinity to the transition state and

stabilizing it, so reducing the activation energy to reach it.

It is important to clarify, however, that the induced fit concept cannot be used to rationalize catalysis. That is, the chemical

catalysis is defined as the reduction of Ea‡ (when the system is already in the ES‡) relative to Ea‡ in the uncatalyzed

reaction in water (without the enzyme). The induced fit only suggests that the barrier is lower in the closed form of the

enzyme but does not tell us what the reason for the barrier reduction is.

Induced fit may be beneficial to the fidelity of molecular recognition in the presence of competition and noise via

the conformational proofreading mechanism .[2]

[edit]Mechanisms of transition state stabilization

These conformational changes also bring catalytic residues in the active site close to the chemical bonds in the substrate

that will be altered in the reaction. After binding takes place, one or more mechanisms of catalysis lowers the energy of the

reaction's transition state, by providing an alternative chemical pathway for the reaction. There are six possible

mechanisms of "over the barrier" catalysis as well as a "through the barrier" mechanism:

[edit]Catalysis by bond strain

This is the principal effect of induced fit binding, where the affinity of the enzyme to the transition state is greater than to

the substrate itself. This induces structural rearrangements which strain substrate bonds into a position closer to the

conformation of the transition state, so lowering the energy difference between the substrate and transition state and

helping catalyze the reaction.

However, the strain effect is, in fact, a ground state destabilization effect, rather than transition state stabilization

effect.[3][4] Furthermore, enzymes are very flexible and they cannot apply large strain effect.[5]

In addition to bond strain in the substrate, bond strain may also be induced within the enzyme itself to activate residues in

the active site.

For example:

Substrate, bound substrate, and transition state conformations of lysozyme.

The substrate, on binding, is distorted from the typical 'chair' hexose ring into the 'sofa' conformation, which is similar in shape to the transition state.

[edit]Catalysis by proximity and orientation

This increases the rate of the reaction as enzyme-substrate interactions align reactive chemical groups and hold them close

together. This reduces the entropy of the reactants and thus makes reactions such as ligations or addition reactions more

favorable, there is a reduction in the overall loss of entropy when two reactants become a single product.

This effect is analogous to an effective increase in concentration of the reagents. The binding of the reagents to the enzyme

gives the reaction intramolecular character, which gives a massive rate increase.

For example:

Similar reactions will occur far faster if the reaction is intramolecular.

The effective concentration of acetate in the intramolecular reaction can be estimated as k2/k1 = 2 x 105 Molar.

However, the situation might be more complex, since modern computational studies have established that traditional

examples of proximity effects cannot be related directly to enzyme entropic effects.[6][7][8] Also, the original entropic

proposal[9] has been found to largely overestimate the contribution of orientation entropy to catalysis.[10]

[edit]Catalysis involving proton donors or acceptors (Acid/Base Catalysis)

See also: Protein pKa calculations

Proton donors and acceptors, i.e. acids and bases, may donate and accept protons in order to stabilize developing charges

in the transition state. This typically has the effect of activatingnucleophile and electrophile groups, or stabilizing leaving

groups. Histidine is often the residue involved in these acid/base reactions, since it has a pKa close to neutral pH and can

therefore both accept and donate protons.

Many reaction mechanisms involving acid/base catalysis assume a substantially altered pKa. This alteration of pKa is

possible through the local environment of the residue.

Conditions Acids Bases

Hydrophobic environment Increase pKa Decrease pKa

Adjacent residues of like charge Increase pKa Decrease pKa

Salt bridge (and hydrogenbond) formation

Decrease pKa Increase pKa

The pKa can be modified significantly by the environment, to the extent that residues which are basic in solution may act

as proton donors, and vice versa.

For example:

Serine protease catalytic mechanism

The initial step of the serine protease catalytic mechanism involves the histidine of the active site accepting a proton from the serine residue. This preparesthe serine as a nucleophile to attack the amide bond of the substrate. This mechanism includes donation of a proton from serine (a base, pKa 14) to

histidine (an acid, pKa 6), made possible due to the local environment of the bases.

It is important to clarify that the modification of the pKa’s is a pure part of the electrostatic mechanism. [4] Furthermore, the

catalytic effect of the above example is mainly associated with the reduction of the pKa of the oxy anion and the increase

in the pKa of the histidine, while the proton transfer from the serine to the histidine is not catalyzed significantly, since it

is not the rate determining barrier.[11]

[edit]Electrostatic catalysis

Stabilization of charged transition states can also be by residues in the active site forming ionic bonds (or partial ionic

charge interactions) with the intermediate. These bonds can either come from acidic or basic side chains found on amino

acids such as lysine, arginine, aspartic acid or glutamic acid or come from metal cofactors such as zinc. Metal ions are

particularly effective and can reduce the pKa of water enough to make it an effective nucleophile.

Systematic computer simulation studies established that electrostatic effects give, by far, the largest contribution to

catalysis.[4] In particular, it has been found that enzyme provides an environment which is more polar than water, and that

the ionic transition states are stabilized by fixed dipoles. This is very different from transition state stabilization in water,

where the water molecules must pay with "reorganization energy".[12] in order to stabilize ionic and charged states. Thus,

the catalysis is associated with the fact that the enzyme polar groups are preorganized[13]

For example:

Carboxypeptidase catalytic mechanism

The tetrahedral intermediate is stabilised by a partial ionic bond between the Zn2+ ion and the negative charge on the oxygen.

[edit]Covalent catalysis

Covalent catalysis involves the substrate forming a transient covalent bond with residues in the active site or with a

cofactor. This adds an additional covalent intermediate to the reaction, and helps to reduce the energy of later transition

states of the reaction. The covalent bond must, at a later stage in the reaction, be broken to regenerate the enzyme. This

mechanism is found in enzymes such as proteases like chymotrypsin and trypsin, where an acyl-enzyme intermediate is

formed. Schiff base formation using the free amine from a lysine residue is another mechanism, as seen in the

enzyme aldolase during glycolysis.

Some enzymes utilize non-amino acid cofactors such as pyridoxal phosphate (PLP) or thiamine pyrophosphate (TPP) to

form covalent intermediates with reactant molecules.[14][15] Such covalent intermediates function to reduce the energy of

later transition states, similar to how covalent intermediates formed with active site amino acid residues allow

stabilization, but the capabilities of cofactors allow enzymes to carryout reactions that amino acid side residues alone

could not. Enzymes utilizing such cofactors include the PLP-dependent enzyme aspartate transaminase and the TPP-

dependent enzyme pyruvate dehydrogenase.[16][17]

It is important to clarify that covalent catalysis does correspond in most cases to simply the use of a specific mechanism

rather than to true catalysis.[4] For example, the energetics of the covalent bond to the serine molecule in chymotrypsin

should be compared to the well-understood covalent bond to the nucleophile in the uncatalyzed solution reaction. A true

proposal of a covalent catalysis (where the barrier is lower than the corresponding barrier in solution) would require, for

example, a partial covalent bond to the transition state by an enzyme group (e.g., a very strong hydrogen bond), and such

effects do not contribute significantly to catalysis.

[edit]Quantum tunneling

These traditional "over the barrier" mechanisms have been challenged in some cases by models and observations of

"through the barrier" mechanisms (quantum tunneling). Some enzymes operate with kinetics which are faster than what

would be predicted by the classical ΔG‡. In "through the barrier" models, a proton or an electron can tunnel through

activation barriers.[18][19]Quantum tunneling for protons has been observed in tryptamine oxidation by aromatic amine

dehydrogenase.[20]

Interestingly, quantum tunneling does not appear to provide a major catalytic advantage, since the tunneling contributions

are similar in the catalyzed and the uncatalyzed reactions in solution.[19][21][22][23] However, the tunneling contribution

(typically enhancing rate constants by a factor of ~1000[20] compared to the rate of reaction for the classical 'over the

barrier' route) is likely crucial to the viability of biological organisms. This emphasizes the general importance of

tunneling reactions in biology.

In 1971-1972 the first quantum-mechanical model of enzyme catalysis was formulated.[24][25]

[edit]Examples of catalytic mechanisms

In reality, most enzyme mechanisms involve a combination of several different types of catalysis.

[edit]Triose phosphate isomerase

Triose phosphate isomerase (EC 5.3.1.1) catalyses the reversible interconvertion of the

two triose phosphates isomers dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate.

[edit]Trypsin

Trypsin (EC 3.4.21.4) is a serine protease that cleaves protein substrates at lysine and arginine amino acid residues.

[edit]Aldolase

Aldolase (EC 4.1.2.13) catalyses the breakdown of fructose 1,6-bisphosphate (F-1,6-BP) into glyceraldehyde 3-

phosphate and dihydroxyacetone phosphate (DHAP).

[edit]

Enzyme assay

Enzyme assayFrom Wikipedia, the free encyclopedia

Beckman DU640 UV/Vis spectrophotometer

Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme

kinetics and enzyme inhibition.

Contents

[hide]

1 Enzyme units

o 1.1 Enzyme activity

o 1.2 Specific activity

o 1.3 Related terminology

2 Types of assay

3 Continuous assays

o 3.1 Spectrophotometric

o 3.2 Fluorometric

o 3.3 Calorimetric

o 3.4 Chemiluminescent

o 3.5 Light Scattering

o 3.6 Microscale Thermophoresis

4 Discontinuous assays

o 4.1 Radiometric

o 4.2 Chromatographic

5 Factors to control in assays

6 List of enzyme assays

7 See also

8 References

9 External links

[edit]Enzyme units

Amounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of

activity, in enzyme units.

[edit]Enzyme activity

Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of

the quantity of active enzyme present and is thus dependent on conditions,which should be specified. The SI unit is

the katal, 1 katal = 1 mol s−1, but this is an excessively large unit. A more practical and commonly used value is 1 enzyme

unit (U) = 1 μmol min−1. 1 U corresponds to 16.67 nanokatals.[1]

Enzyme activity as given in katal generally refers to that of the assumed natural target substrate of the enzyme. Enzyme

activity can also be given as that of certain standardized substrates, such as gelatin, then measured in gelatin digesting

units (GDU), or milk proteins, then measured in milk clotting units (MCU). The units GDU and MCU are based on how

fast one gram of the enzyme will digest gelatin or milk proteins, respectively. 1 GDU equals approximately 1.5 MCU.[2]

[edit]Specific activity

The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein

(expressed in μmol min−1mg−1). Specific activity gives a measurement of the activity of the enzyme. It is the amount of

product formed by an enzyme in a given amount of time under given conditions per milligram of total protein. Specific

activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of total protein. The SI

unit is katal kg−1, but a more practical unit is μmol mg−1 min−1. Specific activity is a measure of enzyme processivity, at a

specific (usually saturating) substrate concentration, and is usually constant for a pure enzyme. For elimination of errors

arising from differences in cultivation batches and/or misfolded enzyme etc. an active site titration needs to be done. This

is a measure of the amount of active enzyme, calculated by e.g. titrating the amount of active sites present by employing

an irreversible inhibitor. The specific activity should then be expressed as μmol min−1 mg−1 active enzyme. If the

molecular weight of the enzyme is known, the turnover number, or μmol product sec−1 μmol−1 of active enzyme, can be

calculated from the specific activity. The turnover number can be visualized as the number of times each enzyme molecule

carries out its catalytic cycle per second.

[edit]Related terminology

The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time (mol

).

The % purity is 100% × (specific activity of enzyme sample / specific activity of pure enzyme). The impure sample has

lower specific activity because some of the mass is not actually enzyme. If the specific activity of 100% pure enzyme is

known, then an impure sample will have a lower specific activity, allowing purity to be calculated.

[edit]Types of assay

All enzyme assays measure either the consumption of substrate or production of product over time. A large number of

different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in

several different ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments:[3]

Initial rate experiments. When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate

intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme

substrate intermediates remains approximately constant over time and the reaction rate changes relatively slowly.

Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the

accumulation of product with time. Because the measurements are carried out for a very short period and because of

the large excess of substrate, the approximation that the amount of free substrate is approximately equal to the

amount of the initial substrate can be made. The initial rate experiment is the simplest to perform and analyze, being

relatively free from complications such as back-reaction and enzyme degradation. It is therefore by far the most

commonly used type of experiment in enzyme kinetics.

Progress curve experiments. In these experiments, the kinetic parameters are determined from expressions for

the species concentrations as a function of time. The concentration of the substrate or product is recorded in time after

the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium. We note in

passing that, while they are less common now, progress curve experiments were widely used in the early period of

enzyme kinetics.

Transient kinetics experiments. In these experiments, reaction behaviour is tracked during the initial fast

transient as the intermediate reaches the steady-state kinetics period. These experiments are more difficult to perform

than either of the above two classes because they require specialist techniques (such as flash photolysis of caged

compounds) or rapid mixing (such as stopped-flow, quenched flow or continuous flow).

Relaxation experiments. In these experiments, an equilibrium mixture of enzyme, substrate and product is

perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored. The

analysis of these experiments requires consideration of the fully reversible reaction. Moreover, relaxation

experiments are relatively insensitive to mechanistic details and are thus not typically used for mechanism

identification, although they can be under appropriate conditions.

Enzyme assays can be split into two groups according to their sampling method: continuous assays, where the assay gives

a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the

concentration of substrates/products determined.

Temperature-controlled cuvette holder in a spectrophotometer.

[edit]Continuous assays

Continuous assays are most convenient, with one assay giving the rate of reaction with no further work necessary. There

are many different types of continuous assays.

[edit]Spectrophotometric

In spectrophotometric assays, you follow the course of the reaction by measuring a change in how much light the assay

solution absorbs. If this light is in the visible region you can actually see a change in the color of the assay, these are

called colorimetric assays. The MTT assay, a redox assay using a tetrazolium dye as substrate is an example of a

colorimetric assay.

UV light is often used, since the common coenzymes NADH and NADPH absorb UV light in their reduced forms, but do

not in their oxidized forms. Anoxidoreductase using NADH as a substrate could therefore be assayed by following the

decrease in UV absorbance at a wavelength of 340 nm as it consumes the coenzyme.[4]

Direct versus coupled assays

Coupled assay for hexokinase using glucose-6-phosphate dehydrogenase.

Even when the enzyme reaction does not result in a change in the absorbance of light, it can still be possible to use a

spectrophotometric assay for the enzyme by using a coupled assay. Here, the product of one reaction is used as the

substrate of another, easily detectable reaction. For example, figure 1 shows the coupled assay for the enzyme hexokinase,

which can be assayed by coupling its production of glucose-6-phosphate to NADPH production, usingglucose-6-

phosphate dehydrogenase.

[edit]Fluorometric

Fluorescence is when a molecule emits light of one wavelength after absorbing light of a different wavelength.

Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction. These

assays are in general much more sensitive than spectrophotometric assays, but can suffer from interference caused by

impurities and the instability of many fluorescent compounds when exposed to light.

An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH. Here, the reduced forms are

fluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be followed by a decrease in

fluorescence and reduction reactions by an increase.[5] Synthetic substrates that release a fluorescent dye in an enzyme-

catalyzed reaction are also available, such as 4-methylumbelliferyl-β-D-galactoside for assaying β-galactosidase.

[edit]Calorimetric

Chemiluminescence of Luminol

Calorimetry is the measurement of the heat released or absorbed by chemical reactions. These assays are very general,

since many reactions involve some change in heat and with use of a microcalorimeter, not much enzyme or substrate is

required. These assays can be used to measure reactions that are impossible to assay in any other way.[6]

[edit]Chemiluminescent

Chemiluminescence is the emission of light by a chemical reaction. Some enzyme reactions produce light and this can be

measured to detect product formation. These types of assay can be extremely sensitive, since the light produced can be

captured by photographic film over days or weeks, but can be hard to quantify, because not all the light released by a

reaction will be detected.

The detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method of detecting

antibodies in western blotting. Another example is the enzyme luciferase, this is found in fireflies and naturally produces

light from its substrate luciferin.

[edit]Light Scattering

Static light scattering measures the product of weight-averaged molar mass and concentration of macromolecules in

solution. Given a fixed total concentration of one or more species over the measurement time, the scattering signal is a

direct measure of the weight-averaged molar mass of the solution, which will vary as complexes form or dissociate. Hence

the measurement quantifies the stoichiometry of the complexes as well as kinetics. Light scattering assays of protein

kinetics is a very general technique that does not require an enzyme.

[edit]Microscale Thermophoresis

Microscale Thermophoresis (MST)[7] measures the size, charge and hydration entropy of molecules/substrates in real

time.[8] The thermophoretic movement of a fluorescently labeled substrate changes significantly as it is modified by an

enzyme. This enzymatic activity can be measured with high time resolution in real time.[9] The material consumption of

the all optical MST method is very low, only 5 µl sample volume and 10nM enzyme concentration are needed to measure

the enzymatic rate constants for activity and inhibition. MST allows to measure the modification of two different

substrates at once (multiplexing) if both substrates are labeled with different fluorophores. Thus substrate competition

experiments can be performed.

[edit]Discontinuous assays

Discontinuous assays are when samples are taken from an enzyme reaction at intervals and the amount of product

production or substrate consumption is measured in these samples.

[edit]Radiometric

Radiometric assays measure the incorporation of radioactivity into substrates or its release from substrates. The radioactive

isotopes most frequently used in these assays are 14C, 32P, 35S and125I. Since radioactive isotopes can allow the specific

labelling of a single atom of a substrate, these assays are both extremely sensitive and specific. They are frequently used in

biochemistry and are often the only way of measuring a specific reaction in crude extracts (the complex mixtures of

enzymes produced when you lyse cells).

Radioactivity is usually measured in these procedures using a scintillation counter.

[edit]Chromatographic

Chromatographic assays measure product formation by separating the reaction mixture into its components

by chromatography. This is usually done by high-performance liquid chromatography(HPLC), but can also use the simpler

technique of thin layer chromatography. Although this approach can need a lot of material, its sensitivity can be increased

by labelling the substrates/products with a radioactive or fluorescent tag. Assay sensitivity has also been increased by

switching protocols to improved chromatographic instruments (e.g. ultra-high pressure liquid chromatography) that

operate at pump pressure a few-fold higher than HPLC instruments (see High-performance liquid

chromatography#Pump_pressure).[10]

[edit]Factors to control in assays

Salt Concentration: Most enzymes cannot tolerate extremely high salt concentrations. The ions interfere with

the weak ionic bonds of proteins. Typical enzymes are active in salt concentrations of 1-500 mM. As usual there are

exceptions such as the halophilic algae and bacteria.

Effects of Temperature: All enzymes work within a range of temperature specific to the organism. Increases in

temperature generally lead to increases in reaction rates. There is a limit to the increase because higher temperatures

lead to a sharp decrease in reaction rates. This is due to the denaturating (alteration) of protein structure resulting

from the breakdown of the weak ionicand hydrogen bonding that stabilize the three dimensional structure of the

enzyme active site.[11] The "optimum" temperature for human enzymes is usually between 35 and 40 °C. The average

temperature for humans is 37 °C. Human enzymes start to denature quickly at temperatures above 40 °C. Enzymes

from thermophilic archaea found in the hot springs are stable up to 100 °C.[12] However, the idea of an "optimum"

rate of an enzyme reaction is misleading, as the rate observed at any temperature is the product of two rates, the

reaction rate and the denaturation rate. If you were to use an assay measuring activity for one second, it would give

high activity at high temperatures, however if you were to use an assay measuring product formation over an hour, it

would give you low activity at these temperatures.

Effects of pH: Most enzymes are sensitive to pH and have specific ranges of activity. All have an optimum pH.

The pH can stop enzyme activity by denaturating (altering) the three dimensional shape of the enzyme by

breaking ionic, and hydrogen bonds. Most enzymes function between a pH of 6 and 8; however pepsin in the stomach

works best at a pH of 2 and trypsin at a pH of 8.

Substrate Saturation: Increasing the substrate concentration increases the rate of reaction (enzyme activity).

However, enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules

are occupied most of the time. At the saturation point, the reaction will not speed up, no matter how much additional

substrate is added. The graph of the reaction rate will plateau.

Level of crowding, large amounts of macromolecules in a solution will alter the rates and equilibrium

constants of enzyme reactions, through an effect called macromolecular crowding.[13]

[edit]List of enzyme assays

MTT assay

Overlay assay

Fluorescein diacetate hydrolysis

[edit]See also

Restriction enzyme

DNase footprinting assay

Enzyme kinetics

[edit]

Protein dynamics

Protein domainFrom Wikipedia, the free encyclopedia

(Redirected from Protein dynamics)

This article may be too long to read and navigate comfortably. Please consider splitting content into sub-articlesand/or condensing it.(November 2010)

Pyruvate kinase, a protein with three domains.

A protein domain is a conserved part of a given protein sequence and structure that can evolve, function, and

exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure

and often can be independently stable and folded. Many proteins consist of several structural domains. One

domain may appear in a variety of different proteins. Molecular evolution uses domains as building blocks and

these may be recombined in different arrangements to create proteins with different functions. Domains vary in

length from between about 25 amino acids up to 500 amino acids in length. The shortest domains such as zinc

fingers are stabilized by metal ions or disulfide bridges. Domains often form functional units, such as the

calcium-binding EF hand domain of calmodulin. Because they are independently stable, domains can be

"swapped" by genetic engineering between one protein and another to make chimeric proteins.

Contents

[hide]

1 Background

2 Domains are units of protein structure

o 2.1 Tertiary structure of domains

o 2.2 Domains have limits on size

o 2.3 Domains and quaternary structure

3 Domains as evolutionary modules

4 Multidomain proteins

o 4.1 Origin

o 4.2 Types of organisation

5 Domains are autonomous folding units

o 5.1 Folding

o 5.2 Advantage of domains in protein folding

6 Domains and protein flexibility

o 6.1 Hinges by secondary structures

o 6.2 Helical to extended conformation

o 6.3 Shear motions

o 6.4 Domain motion and functional dynamics in enzymes

7 Domain definition from structural co-ordinates

o 7.1 Methods

o 7.2 Example domains

8 See also

9 References

10 Key papers

11 External links

o 11.1 Structural domain databases

o 11.2 Sequence domain databases

[edit]Background

The concept of the domain was first proposed in 1973 by Wetlaufer after X-ray crystallographic studies of

hen lysozyme [1] and papain [2] and by limited proteolysis studies ofimmunoglobulins.[3][4] Wetlaufer defined

domains as stable units of protein structure that could fold autonomously. In the past domains have been

described as units of:

compact structure[5]

function and evolution[6]

folding.[7]

Each definition is valid and will often overlap, i.e. a compact structural domain that is found amongst diverse

proteins is likely to fold independently within its structural environment. Nature often brings several domains

together to form multidomain and multifunctional proteins with a vast number of possibilities. [8] In a multidomain

protein, each domain may fulfill its own function independently, or in a concerted manner with its neighbours.

Domains can either serve as modules for building up large assemblies such as virus particles or muscle fibres,

or can provide specific catalytic or binding sites as found in enzymes or regulatory proteins.

An appropriate example is pyruvate kinase, a glycolytic enzyme that plays an important role in regulating the

flux from fructose-1,6-biphosphate to pyruvate. It contains an all-β regulatory domain, an α/β-substrate binding

domain and an α/β-nucleotide binding domain, connected by several polypeptide linkers [9] (see figure, right).

Each domain in this protein occurs in diverse sets of protein families.

The central α/β-barrel substrate binding domain is one of the most common enzyme folds. It is seen in many

different enzyme families catalysing completely unrelated reactions.[10] The α/β-barrel is commonly called

the TIM barrel named after triose phosphate isomerase, which was the first such structure to be solved. [11] It is

currently classified into 26 homologous families in the CATH domain database. [12] The TIM barrel is formed

from a sequence of β-α-β motifs closed by the first and last strand hydrogen bonding together, forming an eight

stranded barrel. There is debate about the evolutionary origin of this domain. One study has suggested that a

single ancestral enzyme could have diverged into several families,[13] while another suggests that a stable TIM-

barrel structure has evolved through convergent evolution.[14]

The TIM-barrel in pyruvate kinase is 'discontinuous', meaning that more than one segment of the polypeptide is

required to form the domain. This is likely to be the result of the insertion of one domain into another during the

protein's evolution. It has been shown from known structures that about a quarter of structural domains are

discontinuous.[15][16] The inserted β-barrel regulatory domain is 'continuous', made up of a single stretch of

polypeptide.

Covalent association of two domains represents a functional and structural advantage since there is an

increase in stability when compared with the same structures non-covalently associated.[17]Other, advantages

are the protection of intermediates within inter-domain enzymatic clefts that may otherwise be unstable in

aqueous environments, and a fixed stoichiometric ratio of the enzymatic activity necessary for a sequential set

of reactions.[18]

[edit]Domains are units of protein structure

Main article: Protein structure

The primary structure (string of amino acids) of a protein ultimately encodes its uniquely folded 3D

conformation.[19] The most important factor governing the folding of a protein into 3D structure is the distribution

of polar and non-polar side chains.[20] Folding is driven by the burial of hydrophobic side chains into the interior

of the molecule so to avoid contact with the aqueous environment. Generally proteins have a core of

hydrophobic residues surrounded by a shell of hydrophilic residues. Since the peptide bonds themselves are

polar they are neutralised by hydrogen bonding with each other when in the hydrophobic environment. This

gives rise to regions of the polypeptide that form regular 3D structural patterns called secondary structure.

There are two main types of secondary structure: α-helices and β-sheets.

Some simple combinations of secondary structure elements have been found to frequently occur in protein

structure and are referred to as supersecondary structure or motifs. For example, the β-hairpin motif consists of

two adjacent antiparallel β-strands joined by a small loop. It is present in most antiparallel β structures both as

an isolated ribbon and as part of more complex β-sheets. Another common super-secondary structure is the β-

α-β motif, which is frequently used to connect two parallel β-strands. The central α-helix connects the C-termini

of the first strand to the N-termini of the second strand, packing its side chains against the β-sheet and

therefore shielding the hydrophobic residues of the β-strands from the surface.

Structural alignment is an important tool for determining domains.

[edit]Tertiary structure of domains

Several motifs pack together to form compact, local, semi-independent units called domains.[5] The overall 3D

structure of the polypeptide chain is referred to as the protein's tertiary structure. Domains are the fundamental

units of tertiary structure, each domain containing an individual hydrophobic core built from secondary

structural units connected by loop regions. The packing of the polypeptide is usually much tighter in the interior

than the exterior of the domain producing a solid-like core and a fluid-like surface.[21] In fact, core residues are

often conserved in a protein family, whereas the residues in loops are less conserved, unless they are involved

in the protein's function. Protein tertiary structure can be divided into four main classes based on the secondary

structural content of the domain.[22]

All-α domains have a domain core built exclusively from α-helices. This class is dominated by small

folds, many of which form a simple bundle with helices running up and down.

All-β domains have a core composed of antiparallel β-sheets, usually two sheets packed against each

other. Various patterns can be identified in the arrangement of the strands, often giving rise to the

identification of recurring motifs, for example the Greek key motif.[23]

α+β domains are a mixture of all-α and all-β motifs. Classification of proteins into this class is difficult

because of overlaps to the other three classes and therefore is not used in the CATHdomain database.[12]

α/β domains are made from a combination of β-α-β motifs that predominantly form a parallel β-sheet

surrounded by amphipathic α-helices. The secondary structures are arranged in layers or barrels.

[edit]Domains have limits on size

Domains have limits on size.[24] The size of individual structural domains varies from 36 residues in E-selectin

to 692 residues in lipoxygenase-1,[15] but the majority, 90%, have less than 200 residues[25] with an average of

approximately 100 residues.[26] Very short domains, less than 40 residues, are often stabilised by metal ions or

disulfide bonds. Larger domains, greater than 300 residues, are likely to consist of multiple hydrophobic

cores.[27]

[edit]Domains and quaternary structure

Many proteins have a quaternary structure, which consists of several polypeptide chains that associate into an

oligomeric molecule. Each polypeptide chain in such a protein is called a subunit. Hemoglobin, for example,

consists of two α and two β subunits. Each of the four chains has an all-α globin fold with a heme pocket.

Domain swapping is a mechanism for forming oligomeric assemblies.[28] In domain swapping, a secondary or

tertiary element of a monomeric protein is replaced by the same element of another protein. Domain swapping

can range from secondary structure elements to whole structural domains. It also represents a model of

evolution for functional adaptation by oligomerisation, e.g. oligomeric enzymes that have their active site at

subunit interfaces.[29]

[edit]Domains as evolutionary modules

Nature is a tinkerer and not an inventor,[30] new sequences are adapted from pre-existing sequences rather

than invented. Domains are the common material used by nature to generate new sequences, they can be

thought of as genetically mobile units, referred to as 'modules'. Often, the C and N termini of domains are close

together in space, allowing them to easily be "slotted into" parent structures during the process of evolution.

Many domain families are found in all three forms of life, Archaea, Bacteria and Eukarya. Domains that are

repeatedly found in diverse proteins are often referred to as modules, examples can be found among

extracellular proteins associated with clotting, fibrinolysis, complement, the extracellular matrix, cell surface

adhesion molecules and cytokine receptors.[31]

Molecular evolution gives rise to families of related proteins with similar sequence and structure. However,

sequence similarities can be extremely low between proteins that share the same structure. Protein structures

may be similar because proteins have diverged from a common ancestor. Alternatively, some folds may be

more favored than others as they represent stable arrangements of secondary structures and some proteins

may converge towards these folds over the course of evolution . There are currently about 45,000

experimentally determined protein 3D structures deposited within the Protein Data Bank (PDB).[32] However this

set contains a lot of identical or very similar structures. All proteins should be classified to structural families to

understand their evolutionary relationships. Structural comparisons are best achieved at the domain level. For

this reason many algorithms have been developed to automatically assign domains in proteins with known 3D

structure, see 'Domain definition from structural co-ordinates'.

The CATH domain database classifies domains into approximately 800 fold families, ten of these folds are

highly populated and are referred to as 'super-folds'. Super-folds are defined as folds for which there are at

least three structures without significant sequence similarity.[33] The most populated is the α/β-barrel super-fold

as described previously.

[edit]Multidomain proteins

The majority of genomic proteins, two-thirds in unicellular organisms and more than 80% in metazoa, are

multidomain proteins created as a result of gene duplication events.[34] Many domains in multidomain structures

could have once existed as independent proteins. More and more domains in eukaryotic multidomain proteins

can be found as independent proteins in prokaryotes.[35] For example, vertebrates have a multi-enzyme

polypeptide containing the GAR synthetase, AIR synthetase and GAR transformylase modules (GARs-AIRs-

GARt; GAR: glycinamide ribonucleotide synthetase/transferase; AIR: aminoimidazole ribonucleotide

synthetase). In insects, the polypeptide appears as GARs-(AIRs)2-GARt, in yeast GARs-AIRs is encoded

separately from GARt, and in bacteria each domain is encoded separately. [36]

[edit]Origin

Multidomain proteins are likely to have emerged from a selective pressure during evolution to create new

functions. Various proteins have diverged from common ancestors by different combinations and associations

of domains. Modular units frequently move about, within and between biological systems through mechanisms

of genetic shuffling:

transposition of mobile elements including horizontal transfers (between species); [37]

gross rearrangements such as inversions, translocations, deletions and duplications;

homologous recombination;

slippage of DNA polymerase during replication.

[edit]Types of organisation

Different insertions of similar PH domainmodules (maroon) into two different proteins.

The simplest multidomain organisation seen in proteins is that of a single domain repeated in tandem. [38] The

domains may interact with each other (domain-domain interaction) or remain isolated, like beads on string. The

giant 30,000 residue muscle protein titin comprises about 120 fibronectin-III-type and Ig-type domains.[39] In the

serine proteases, a gene duplication event has led to the formation of a two β-barrel domain enzyme.[40] The

repeats have diverged so widely that there is no obvious sequence similarity between them. The active site is

located at a cleft between the two β-barrel domains, in which functionally important residues are contributed

from each domain. Genetically engineered mutants of the chymotrypsin serine protease were shown to have

some proteinase activity even though their active site residues were abolished and it has therefore been

postulated that the duplication event enhanced the enzyme's activity. [40]

Modules frequently display different connectivity relationships, as illustrated by the kinesins and ABC

transporters. The kinesin motor domain can be at either end of a polypeptide chain that includes a coiled-coil

region and a cargo domain.[41] ABC transporters are built with up to four domains consisting of two unrelated

modules, ATP-binding cassette and an integral membrane module, arranged in various combinations.

Not only do domains recombine, but there are many examples of a domain having been inserted into another.

Sequence or structural similarities to other domains demonstrate that homologues of inserted and parent

domains can exist independently. An example is that of the 'fingers' inserted into the 'palm' domain within the

polymerases of the Pol I family.[42] Since a domain can be inserted into another, there should always be at least

one continuous domain in a multidomain protein. This is the main difference between definitions of structural

domains and evolutionary/functional domains. An evolutionary domain will be limited to one or two connections

between domains, whereas structural domains can have unlimited connections, within a given criterion of the

existence of a common core. Several structural domains could be assigned to an evolutionary domain.

[edit]Domains are autonomous folding units

[edit]Folding

Further information: Protein folding

Protein folding - the unsolved problem : Since the seminal work of Anfinsen over forty years ago,[19] the goal

to completely understand the mechanism by which a polypeptide rapidly folds into its stable native

conformation remains elusive. Many experimental folding studies have contributed much to our understanding,

but the principles that govern protein folding are still based on those discovered in the very first studies of

folding. Anfinsen showed that the native state of a protein is thermodynamically stable, the conformation being

at a global minimum of its free energy.

Folding is a directed search of conformational space allowing the protein to fold on a biologically feasible time

scale. The Levinthal paradox states that if an averaged sized protein would sample all possible conformations

before finding the one with the lowest energy, the whole process would take billions of years. [43] Proteins

typically fold within 0.1 and 1000 seconds. Therefore, the protein folding process must be directed some way

through a specific folding pathway. The forces that direct this search are likely to be a combination of local and

global influences whose effects are felt at various stages of the reaction. [44]

Advances in experimental and theoretical studies have shown that folding can be viewed in terms of energy

landscapes,[45][46] where folding kinetics is considered as a progressive organisation of an ensemble of partially

folded structures through which a protein passes on its way to the folded structure. This has been described in

terms of a folding funnel, in which an unfolded protein has a large number of conformational states available

and there are fewer states available to the folded protein. A funnel implies that for protein folding there is a

decrease in energy and loss of entropy with increasing tertiary structure formation. The local roughness of the

funnel reflects kinetic traps, corresponding to the accumulation of misfolded intermediates. A folding chain

progresses toward lower intra-chain free-energies by increasing its compactness. The chains conformational

options become increasingly narrowed ultimately toward one native structure.

[edit]Advantage of domains in protein folding

The organisation of large proteins by structural domains represents an advantage for protein folding, with each

domain being able to individually fold, accelerating the folding process and reducing a potentially large

combination of residue interactions. Furthermore, given the observed random distribution of hydrophobic

residues in proteins,[47] domain formation appears to be the optimal solution for a large protein to bury its

hydrophobic residues while keeping the hydrophilic residues at the surface.[48][49]

However, the role of inter-domain interactions in protein folding and in energetics of stabilisation of the native

structure, probably differs for each protein. In T4 lysozyme, the influence of one domain on the other is so

strong that the entire molecule is resistant to proteolytic cleavage. In this case, folding is a sequential process

where the C-terminal domain is required to fold independently in an early step, and the other domain requires

the presence of the folded C-terminal domain for folding and stabilisation.[50]

It has been found that the folding of an isolated domain can take place at the same rate or sometimes faster

than that of the integrated domain,[51] suggesting that unfavourable interactions with the rest of the protein can

occur during folding. Several arguments suggest that the slowest step in the folding of large proteins is the

pairing of the folded domains.[27] This is either because the domains are not folded entirely correctly or because

the small adjustments required for their interaction are energetically unfavourable, [52] such as the removal of

water from the domain interface.

[edit]Domains and protein flexibility

The presence of multiple domains in proteins gives rise to a great deal of flexibility and mobility, leading

to protein domain dynamics.[53] Domain motions can be inferred by comparing different structures of a protein

(as in Database of Molecular Motions), or they can be directly observed using spectra[54][55] measured

by neutron spin echo spectroscopy. They can also be suggested by sampling in extensive molecular dynamics

trajectories.[56] Domain motions are important for:[57]

catalysis[citation needed]

regulatory activity

transport of metabolites

formation of protein assemblies

cellular locomotion

One of the largest observed domain motions is the `swivelling' mechanism in pyruvate phosphate dikinase. The

phosphoinositide domain swivels between two states in order to bring a phosphate group from the active site of

the nucleotide binding domain to that of the phosphoenolpyruvate/pyruvate domain. [58] The phosphate group is

moved over a distance of 45A involving a domain motion of about 100 degrees around a single residue. In

enzymes, the closure of one domain onto another captures a substrate by an induced fit, allowing the reaction

to take place in a controlled way. A detailed analysis by Gerstein led to the classification of two basic types of

domain motion; hinge and shear.[57] Only a relatively small portion of the chain, namely the inter-domain linker

and side chains undergo significant conformational changes upon domain rearrangement. [59]

[edit]Hinges by secondary structures

A study by Hayward[60] found that the termini of α-helices and β-sheets form hinges in a large number of cases.

Many hinges were found to involve two secondary structure elements acting like hinges of a door, allowing an

opening and closing motion to occur. This can arise when two neighbouring strands within a β-sheet situated in

one domain, diverge apart as they join the other domain. The two resulting termini then form the bending

regions between the two domains. α-helices that preserve their hydrogen bonding network when bent are

found to behave as mechanical hinges, storing `elastic energy' that drives the closure of domains for rapid

capture of a substrate.[60]

[edit]Helical to extended conformation

The interconversion of helical and extended conformations at the site of a domain boundary is not uncommon.

In calmodulin, torsion angles change for five residues in the middle of a domain linking α-helix. The helix is split

into two, almost perpendicular, smaller helices separated by four residues of an extended strand. [61][62]

[edit]Shear motions

Shear motions involve a small sliding movement of domain interfaces, controlled by the amino acid side chains

within the interface. Proteins displaying shear motions often have a layered architecture: stacking of secondary

structures. The interdomain linker has merely the role of keeping the domains in close proximity.

[edit]Domain motion and functional dynamics in enzymes

The analysis of the internal dynamics of structurally different, but functionally similar enzymes has highlighted a

common relationship between the positioning of the active site and the two principal protein sub-domains. In

fact, for several members of the hydrolase superfamily, the catalytic site is located close to the interface

separating the two principal quasi-rigid domains.[56] Such positioning appears instrumental for maintaining the

precise geometry of the active site, while allowing for an appreciable functionally oriented modulation of the

flanking regions resulting from the relative motion of the two sub-domains.

[edit]Domain definition from structural co-ordinates

The importance of domains as structural building blocks and elements of evolution has brought about many

automated methods for their identification and classification in proteins of known structure. Automatic

procedures for reliable domain assignment is essential for the generation of the domain databases, especially

as the number of protein structures is increasing. Although the boundaries of a domain can be determined by

visual inspection, construction of an automated method is not straightforward. Problems occur when faced with

domains that are discontinuous or highly associated.[63] The fact that there is no standard definition of what a

domain really is has meant that domain assignments have varied enormously, with each researcher using a

unique set of criteria.[64]

A structural domain is a compact, globular sub-structure with more interactions within it than with the rest of the

protein.[59] Therefore, a structural domain can be determined by two visual characteristics; its compactness and

its extent of isolation.[65] Measures of local compactness in proteins have been used in many of the early

methods of domain assignment[66][67][68][69] and in several of the more recent methods.[25][70][71][72][73]

[edit]Methods

One of the first algorithms[66] used a Cα-Cα distance map together with a hierarchical clustering routine that

considered proteins as several small segments, 10 residues in length. The initial segments were clustered one

after another based on inter-segment distances; segments with the shortest distances were clustered and

considered as single segments thereafter. The stepwise clustering finally included the full protein. Go[69] also

exploited the fact that inter-domain distances are normally larger than intra-domain distances; all possible Cα-

Cα distances were represented as diagonal plots in which there were distinct patterns for helices, extended

strands and combinations of secondary structures.

The method by Sowdhamini and Blundell clusters secondary structures in a protein based on their Cα-Cα

distances and identifies domains from the pattern in their dendrograms.[63] As the procedure does not consider

the protein as a continuous chain of amino acids there are no problems in treating discontinuous domains.

Specific nodes in these dendrograms are identified as tertiary structural clusters of the protein, these include

both super-secondary structures and domains. The DOMAK algorithm is used to create the 3Dee domain

database.[71] It calculates a 'split value' from the number of each type of contact when the protein is divided

arbitrarily into two parts. This split value is large when the two parts of the structure are distinct.

The method of Wodak and Janin[74] was based on the calculated interface areas between two chain segments

repeatedly cleaved at various residue positions. Interface areas were calculated by comparing surface areas of

the cleaved segments with that of the native structure. Potential domain boundaries can be identified at a site

where the interface area was at a minimum. Other methods have used measures of solvent accessibility to

calculate compactness.[25][75][76]

The PUU algorithm[16] incorporates a harmonic model used to approximate inter-domain dynamics. The

underlying physical concept is that many rigid interactions will occur within each domain and loose interactions

will occur between domains. This algorithm is used to define domains in the FSSP domain database.[70]

Swindells (1995) developed a method, DETECTIVE, for identification of domains in protein structures based on

the idea that domains have a hydrophobic interior. Deficiencies were found to occur when hydrophobic cores

from different domains continue through the interface region.

RigidFinder is a novel method for identification of protein rigid blocks (domains and loops) from two different

conformations. Rigid blocks are defined as blocks where all inter residue distances are conserved across

conformations.

A general method to identify dynamical domains, that is protein regions that behave approximately as rigid

units in the course of structural fluctuations, has been introduced by Potestio et al.[56]and, among other

applications was also used to compare the consistency of the dynamics-based domain subdivisions with

standard structure-based ones. The method, termed PiSQRD, is publicly available in the form of a

webserver.[77] The latter allows users to optimally subdivide single-chain or multimeric proteins into quasi-rigid

domains[56][77] based on the collective modes of fluctuation of the system. By default the latter are calculated

through an elastic network model;[78] alternatively pre-calculated essential dynamical spaces can be uploaded

by the user.

[edit]Example domains

Armadillo repeats : named after the β-catenin-like Armadillo protein of the fruit fly Drosophila.

Basic Leucine zipper domain (bZIP domain) : is found in many DNA-binding eukaryotic proteins. One

part of the domain contains a region that mediates sequence-specific DNA-binding properties and the

Leucine zipper that is required for the dimerization of two DNA-binding regions. The DNA-binding region

comprises a number of basic aminoacids such as arginine and lysine

Cadherin repeats : Cadherins function as Ca2+-dependent cell-cell adhesion proteins. Cadherin

domains are extracellular regions which mediate cell-to-cell homophilic binding between cadherins on the

surface of adjacent cells.

Death effector domain (DED) : allows protein-protein binding by homotypic interactions (DED-

DED). Caspase proteases trigger apoptosis via proteolytic cascades. Pro-Caspase-8 and pro-caspase-9

bind to specific adaptor molecules via DED domains and this leads to autoactivation of caspases.

EF hand : a helix-turn-helix structural motif found in each structural domain of the signaling

protein calmodulin and in the muscle protein troponin-C.

Immunoglobulin-like domains : are found in proteins of the immunoglobulin superfamily (IgSF).[79] They

contain about 70-110 amino acids and are classified into different categories (IgV, IgC1, IgC2 and IgI)

according to their size and function. They possess a characteristic fold in which two beta sheets form a

"sandwich" that is stabilized by interactions between conserved cysteinesand other charged amino acids.

They are important for protein-to-protein interactions in processes of cell adhesion, cell activation, and

molecular recognition. These domains are commonly found in molecules with roles in the immune system.

Phosphotyrosine-binding domain (PTB) : PTB domains usually bind to phosphorylated tyrosine

residues. They are often found in signal transduction proteins. PTB-domain binding specificity is

determined by residues to the amino-terminal side of the phosphotyrosine. Examples: the PTB domains of

both SHC and IRS-1 bind to a NPXpY sequence. PTB-containing proteins such as SHC and IRS-1 are

important for insulin responses of human cells.

Pleckstrin homology domain (PH) : PH domains bind phosphoinositides with high affinity. Specificity

for PtdIns(3)P, PtdIns(4)P, PtdIns(3,4)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 have all been observed.

Given the fact that phosphoinositides are sequestered to various cell membranes (due to their long

lipophilic tail) the PH domains usually causes recruitment of the protein in question to a membrane where

the protein can exert a certain function in cell signalling, cytoskeletal reorganization or membrane

trafficking.

Src homology 2 domain (SH2) : SH2 domains are often found in signal transduction proteins. SH2

domains confer binding to phosphorylated tyrosine (pTyr). Named after the phosphotyrosine binding

domain of the src viral oncogene, which is itself a tyrosine kinase. See also: SH3 domain.

Zinc finger DNA binding domain (ZnF_GATA) : ZnF_GATA domain-containing proteins are

typically transcription factors that usually bind to the DNA sequence [AT]GATA[AG] of promoters.

[edit]See also

Binding domain

Short linear motif

Protein

Protein structure

Protein structure prediction

Protein structure prediction software

Protein family

Structural biology

Structural Classification of Proteins (SCOP)

CATH

The Proteolysis Map

The Proteolysis MapFrom Wikipedia, the free encyclopedia

PMAP logo

The Proteolysis MAP (PMAP) is an integrated web resource focused on proteases.[1]

Contents

[hide]

1 Rationale

2 History and

funding

3 Focal point

4 The goal

5 Database

content

6 Usage

7 See also

8 References

9 External

links

[edit]Rationale

PMAP is to aid the protease researchers in reasoning about proteolytic networks and metabolic pathways.

[edit]History and funding

PMAP was originally created at the Burnham Institute for Medical Research, La Jolla, California. In 2004

the National Institutes of Health (NIH) selected a team led by Jeffrey W. Smith, to establish the Center on

Proteolytic Pathways (CPP). As part of the NIH Roadmap for Biomedical research, the center develops

technology to study the behavior of proteins and to disburse that knowledge to the scientific community at

large.

[edit]Focal point

Proteases are a class of enzymes that regulate much of what happens in the human body, both inside

the cell and out, by cleaving peptide bonds in proteins. Through this activity, they govern the four essential cell

functions: differentiation, motility, division and cell death — and activate important extracellular episodes, such

as the biochemical cascade effect in blood clotting. Simply stated, life could not exist without them. Extensive

on-line classification system for proteases (also referred as peptidases) is deposited in the MEROPS database.

[edit]The goal

Proteolytic pathways, or proteolysis, are the series of events controlled by proteases that occur in response to

specific stimuli. In addition to the clotting of blood, the production of insulin can be viewed as a proteolytic

pathway, as the activation, regulation and inhibition of that protein is the result of proteases reacting to

changing glucose levels and triggering other proteases downstream.

[edit]Database content

PMAP integrates five databases (DBs), linked together in one environment. (1)ProteaseDB and

(2)SubstrateDB, are driven by an automated annotation pipeline that generates dynamic ‘Molecule Pages’, rich

in molecular information. (3)CutDB[2] has information on more than 6,600 proteolytic events, and (4)ProfileDB is

dedicated to information of the substrate recognition specificity of proteases. (5)PathwayDB, just begun

accumulation of metabolic pathways whose function can be dynamically modeled in a rule-based manner.

Hypothetical networks are inferred by semi-automated culling of the literature. Additionally, protease software

tools are available for the analysis of individual proteases and proteome-wide data sets.

[edit]Usage

Popular destinations in PMAP are Protease Molecule Pages and Substrate Molecule Pages. Protease

Molecule Pages show recent news in PubMed literature of the protease, known proteolytic events, protein

domain location and protein structure view, as well as a cross annotation in other bioinformatic databases

section. Substrate Molecule Pages display protein domains and experimentally derived protease cut-sites for a

given protein target of interest.

[edit]See also

Metabolic pathway

Cytoscape

Computational genomics

Metabolic network modelling

Protein-protein interaction prediction

MEROPS

[edit]References

1. ^ Igarashi Y, Heureux E, Doctor KS, Talwar P, Gramatikova S, Gramatikoff K, Zhang Y, Blinov M, Ibragimova SS, Boyd S,

Ratnikov B, Cieplak P, Godzik A, Smith JW, Osterman AL, Eroshkin AM. PMAP: databases for analyzing proteolytic events and

pathways. Nucleic Acids Research. 2008 Oct 8.[Epub ahead of print]

2. ^ Igarashi Y, Eroshkin A, Gramatikova S, Gramatikoff K, Zhang Y, Smith JW, Osterman AL, Godzik A. CutDB: a proteolytic

event database. Nucleic Acids Research. 2007 D546-9

[edit]External links

Official website

Host institution website

Proteolysis Cut Site database - curated expert annotation from users

Protease cut sites graphical interface

Protease cutting predictor

List of proteases and their specificities

International Proteolysis Society

Merops - the peptidase database

List of protease inhibitors

Proteases at the US National Library of Medicine Medical Subject Headings (MeSH)

RNA Biocatalysis

RibozymeFrom Wikipedia, the free encyclopedia

(Redirected from RNA Biocatalysis)

This article is about the chemical. For the rock band, see Ribozyme (band).

Structure of hammerhead ribozyme

A ribozyme is an RNA molecule with a well defined tertiary structure that enables it to perform a chemical

reaction. Many ribozymes are catalytic, but some such as self-cleaving ribozymes are consumed by their

reactions. Ribozyme means ribonucleic acid enzyme. It may also be called an RNA enzyme or catalytic RNA.

Many natural ribozymes cleave one of their own phosphodiester bonds (self-cleaving ribozymes), or bonds in

other RNAs. Some have been found to catalyze the aminotransferase activity of the ribosome. Examples of

ribozymes include the hammerhead ribozyme, the VS ribozyme and the hairpin ribozyme.

Investigators studying the origin of life have produced ribozymes in the laboratory that are capable of catalyzing

their own synthesis under very specific conditions, such as an RNA polymerase ribozyme.[1] Mutagenesis and

selection has been performed resulting in isolation of improved variants of the "Round-18" polymerase

ribozyme from 2001. "B6.61" is able to add up to 20 nucleotides to a primer template in 24 hours, until it

decomposes by cleavage of its phosphodiester bonds.[2] The "tC19Z" ribozyme can add up to

95 nucleotides with a fidelity of 0.0083 mutations/nucleotide.[3]

Some ribozymes may play an important role as therapeutic agents, as enzymes which tailor defined RNA

sequences, as biosensors, and for applications infunctional genomics and gene discovery.[4]

Contents

[hide]

1 Discovery

2 Activity

3 Known

ribozymes

4 Artificial

ribozymes

5 Applications

6 See also

7 References

8 Further

reading

9 External

links

[edit]Discovery

Schematic showing ribozyme cleavage of RNA.

Before the discovery of ribozymes, enzymes, which are defined as catalytic proteins,[5] were the only known

biological catalysts. In 1967,Carl Woese, Francis Crick, and Leslie Orgel were the first to suggest that RNA

could act as a catalyst. This idea was based upon the discovery that RNA can form complex secondary

structures.[6] The first ribozymes were discovered in the 1980s by Thomas R. Cech, who was studying

RNA splicing in the ciliated protozoan Tetrahymena thermophila and Sidney Altman, who was working on the

bacterialRNase P complex. These ribozymes were found in the intron of an RNA transcript, which removed

itself from the transcript, as well as in the RNA component of the RNase P complex, which is involved in the

maturation of pre-tRNAs. In 1989, Thomas R. Cech and Sidney Altman won the Nobel Prize in chemistry for

their "discovery of catalytic properties of RNA."[7] The term ribozyme was first introduced by Kelly Kruger et

al. in 1982 in a paper published in Cell.[8]

It had been a firmly established belief in biology that catalysis was reserved for proteins. In retrospect, catalytic

RNA makes a lot of sense. This is based on the old question regarding the origin of life: Which comes first,

enzymes that do the work of the cell or nucleic acids that carry the information required to produce the

enzymes? Ribo-Nucleic acids as catalysts circumvents this problem. RNA, in essence can be both the chicken

and the egg.[9]

In the 1970s Thomas Cech, at the University of Colorado at Boulder, was studying the excision of introns in a

ribosomal RNA gene in Tetrahymena thermophila. While trying to purify the enzyme responsible for splicing

reaction, he found that intron could be spliced out in the absence of any added cell extract. As much as they

tried, Cech and his colleagues could not identify any protein associated with the splicing reaction. After much

work, Cech proposed that the intron sequence portion of the RNA could break and

reform phosphodiester bonds. At about the same time, Sidney Altman, a professor at Yale University, was

studying the way tRNA molecules are processed in the cell when he and his colleagues isolated an enzyme

called RNase-P, which is responsible for conversion of a precursor tRNA into the active tRNA. Much to their

surprise, they found that RNase-P contained RNA in addition to protein and that RNA was an essential

component of the active enzyme. This was such a foreign idea that they had difficulty publishing their findings.

The following year, Altman demonstrated that RNA can act as a catalyst by showing that the RNase-P RNA

subunit could catalyze the cleavage of precursor tRNA into active tRNA in the absence of any protein

component.

Since Cech's and Altman's discovery, other investigators have discovered other examples of self-cleaving RNA

or catalytic RNA molecules. Many ribozymes have either a hairpin – or hammerhead – shaped active center

and a unique secondary structure that allows them to cleave other RNA molecules at specific sequences. It is

now possible to make ribozymes that will specifically cleave any RNA molecule. These RNA catalysts may

have pharmaceutical applications. For example, a ribozyme has been designed to cleave the RNA of HIV. If

such a ribozyme was made by a cell, all incoming virus particles would have their RNA genome cleaved by the

ribozyme, which would prevent infection.

[edit]Activity

Although most ribozymes are quite rare in the cell, their roles are sometimes essential to life. For example, the

functional part of the ribosome, the molecular machine that translates RNA into proteins, is fundamentally a

ribozyme, composed of RNA tertiary structural motifs that are often coordinated to metal ions such

as Mg2+ as cofactors. There is no requirement for divalent cationsin a five-nucleotide RNA that can

catalyze trans-phenylalanation of a four-nucleotide substrate which has three base complementary sequence

with the catalyst. The catalyst and substrate were devised by truncation of the C3 ribozyme.[10]

RNA can also act as a hereditary molecule, which encouraged Walter Gilbert to propose that in the distant

past, the cell used RNA as both the genetic material and the structural and catalytic molecule, rather than

dividing these functions between DNA and protein as they are today. This hypothesis became known as the

"RNA world hypothesis" of the origin of life.

If ribozymes were the first molecular machines used by early life, then today's remaining ribozymes—such as

the ribosome machinery—could be considered living fossils of a life based primarily on nucleic acids.

A recent test-tube study of prion folding suggests that an RNA may catalyze the pathological protein

conformation in the manner of a chaperone enzyme.[11]

Ribozymes have been shown to be involved in the viral concatemer cleavage that precedes the packing of viral

genetic material into virions.[12][13]

[edit]Known ribozymes

Naturally occurring ribozymes include:

Peptidyl transferase 23S rRNA - Found in all living cells

RNase P

Group I and Group II introns

GIR1 branching ribozyme[14]

Leadzyme - Although initially created in vitro, natural examples have been found

Hairpin ribozyme

Hammerhead ribozyme

HDV ribozyme

Mammalian CPEB3 ribozyme

VS ribozyme

glmS ribozyme

CoTC ribozyme

[edit]Artificial ribozymes

Since the discovery of ribozymes that exist in living organisms, there has been interest in the study of new

synthetic ribozymes made in the laboratory. For example, artificially-produced self-cleaving RNAs that have

good enzymatic activity have been produced. Tang and Breaker[15] isolated self-cleaving RNAs by in vitro

selection of RNAs originating from random-sequence RNAs. Some of the synthetic ribozymes that were

produced had novel structures, while some were similar to the naturally occurring hammerhead ribozyme.

The techniques used to create artificial ribozymes involve Darwinian evolution. This approach takes advantage

of RNA's dual nature as both a catalyst and an informational polymer, making it easy for an investigator to

produce vast populations of RNA catalysts using polymerase enzymes. The ribozymes are mutated by reverse

transcribing them with reverse transcriptase into various cDNAand amplified with mutagenic PCR. The

selection parameters in these experiments often differ. One approach for selecting a ligase ribozyme involves

using biotin tags, which are covalently linked to the substrate. If a molecule possesses the

desired ligase activity, a streptavidin matrix can be used to recover the active molecules.

Lincoln and Joyce developed an RNA enzyme system capable of self replication in about an hour. By utilizing

molecular competition (in vitro evolution) of a candidate enzyme mixture, a pair of RNA enzymes emerged, in

which each synthesizes the other from synthetic oligonucleotides, with no protein present. [16]

[edit]Applications

A type of synthetic ribozyme directed against HIV RNA called gene shears has been developed and has

entered clinical testing for HIV infection.[17][18]

[edit]See also

Deoxyribozyme

Spiegelman Monster

Catalysis

Enzyme

RNA world hypothesis

Peptide nucleic acid

Nucleic acid analogues

PAH world hypothesis

SELEX

OLE RNA

SUMO enzymes

SUMO enzymesFrom Wikipedia, the free encyclopedia

SUMO enzymatic cascade

SUMO enzymatic cascade catalyzes the dynamic posttranslational modification process of sumoylation (i.e.

transfer of SUMO protein to other proteins). The Small Ubiquitin-related Modifier, SUMO-1,[1][2] is a ubiquitin-like

family member that is conjugated to its substrates through three discrete enzymatic steps (see the figure on the

right): activation, involving the E1 enzyme (SAE1/SAE2);[3] conjugation, involving the E2 enzyme

(UBC9);[4][5] substrate modification, through the cooperation of the E2 and E3 [6] protein ligases.[7]

SUMO pathway modifies hundreds of proteins that participate in diverse cellular processes. [8] SUMO pathway

is the most studied ubiquitin-like pathway that regulates a wide range of cellular events,[9]evidenced by a large

number of sumoylated proteins identified in more than ten large-scale studies.[10][11][12][13][14][15][16][17][18][19][20]

[edit]See also

Metabolism

Metabolic network

Metabolic network modelling

Ki Database

Ki DatabaseFrom Wikipedia, the free encyclopedia

NIMH PDSP

Headquarters Chapel Hill, North CarolinaAugust, 2006

Key people Director: Bryan Roth MD, PhD

Website PDSP home page

The Ki Database (or Ki DB) is a public domain database of published binding affinities (Ki)

of drugs and chemical compounds for receptors,neurotransmitter transporters, ion channels, and enzymes. The

resource is maintained by the University of North Carolina at Chapel Hill and is funded by

the NIMH Psychoactive Drug Screening Program and by a gift from the Heffter Research Institute. As of April

2010, the database had data for 7449 compounds at 738 different receptors and, as of 31 January 2012,

55472 Ki values.

[edit]External links

Description

Search form

BindingDB.org - A similar publicly available database

This pharmacology-related article is a stub. You can help Wikipedia by expanding it.

Proteonomics and protein engineering

ProteomicsFrom Wikipedia, the free encyclopedia

(Redirected from Proteonomics)

For the journal Proteomics, see Proteomics (journal).

Robotic preparation of MALDI mass spectrometry samples on a sample carrier.

Proteomics is the large-scale study of proteins, particularly their structures and functions.[1][2] Proteins are vital

parts of living organisms, as they are the main components of the physiological metabolic pathways of cells.

The term "proteomics" was first coined in 1997[3] to make an analogy with genomics, the study of the genes.

The word "proteome" is a blend of "protein" and "genome", and was coined byMarc Wilkins in 1994 while

working on the concept as a PhD student.[4][5] The proteome is the entire complement of proteins,[4] including

the modifications made to a particular set of proteins, produced by an organism or system. This will vary with

time and distinct requirements, or stresses, that a cell or organism undergoes.

While proteomics generally refers to the large-scale experimental analysis of proteins, it is often specifically

used for protein purification and mass spectrometry.

Contents

[hide]

1 Complexity of the problem

o 1.1 Post-translational modifications

1.1.1 Phosphorylation

1.1.2 Ubiquitination

1.1.3 Additional modifications

o 1.2 Distinct proteins are made under distinct settings

2 Limitations of genomics and proteomics studies

3 Methods of studying proteins

o 3.1 Identifying proteins that are post-translationally modified

o 3.2 Determining the existence of proteins in complex mixtures

o 3.3 Computational methods in studying protein biomarkers

4 Establishing protein–protein interactions

5 Practical applications of proteomics

o 5.1 Biomarkers

o 5.2 Proteogenomics

o 5.3 Current research methodologies

6 See also

o 6.1 Protein databases

o 6.2 Research centers

7 References

8 Bibliography

9 External links

[edit]Complexity of the problem

After genomics and transcriptomics, proteomics is considered the next step in the study of biological systems. It

is much more complicated than genomics mostly because while an organism'sgenome is more or less

constant, the proteome differs from cell to cell and from time to time. This is because distinct genes are

expressed in distinct cell types. This means that even the basic set of proteins which are produced in a cell

needs to be determined.

In the past this was done by mRNA analysis, but this was found not to correlate with protein content. [6][7] It is

now known that mRNA is not always translated into protein,[8] and the amount of protein produced for a given

amount of mRNA depends on the gene it is transcribed from and on the current physiological state of the cell.

Proteomics confirms the presence of the protein and provides a direct measure of the quantity present.

[edit]Post-translational modifications

Not only does the translation from mRNA cause differences, but many proteins are also subjected to a wide

variety of chemical modifications after translation. Many of these post-translational modifications are critical to

the protein's function.

[edit]Phosphorylation

One such modification is phosphorylation, which happens to many enzymes and structural proteins in the

process of cell signaling. The addition of a phosphate to particular amino acids—most

commonly serine and threonine[9] mediated by serine/threonine kinases, or more rarely tyrosine mediated by

tyrosine kinases—causes a protein to become a target for binding or interacting with a distinct set of other

proteins that recognize the phosphorylated domain.

Because protein phosphorylation is one of the most-studied protein modifications, many "proteomic" efforts are

geared to determining the set of phosphorylated proteins in a particular cell or tissue-type under particular

circumstances. This alerts the scientist to the signaling pathways that may be active in that instance.

[edit]Ubiquitination

Ubiquitin is a small protein that can be affixed to certain protein substrates by enzymes called E3 ubiquitin

ligases. Determining which proteins are poly-ubiquitinated can be helpful in understanding how protein

pathways are regulated. This is therefore an additional legitimate "proteomic" study. Similarly, once it is

determined which substrates are ubiquitinated by each ligase, determining the set of ligases expressed in a

particular cell type will be helpful.

[edit]Additional modifications

Listing all the protein modifications that might be studied in a "Proteomics" project would require a discussion of

most of biochemistry; therefore, a short list will serve here to illustrate the complexity of the problem. In addition

to phosphorylation and ubiquitination, proteins can be subjected to (among

others) methylation, acetylation, glycosylation, oxidation and nitrosylation. Some proteins undergo ALL of these

modifications, often in time-dependent combinations, aptly illustrating the potential complexity one has to deal

with when studying protein structure and function.

[edit]Distinct proteins are made under distinct settings

Even if one is studying a particular cell type, that cell may make different sets of proteins at different times, or

under different conditions. Furthermore, as mentioned, any one protein can undergo a wide range of post-

translational modifications.

Therefore a "proteomics" study can become quite complex very quickly, even if the object of the study is very

restricted. In more ambitious settings, such as when a biomarker for a tumor is sought – when the proteomics

scientist is obliged to study sera samples from multiple cancer patients – the amount of complexity that must be

dealt with is as great as in any modern biological project.

[edit]Limitations of genomics and proteomics studies

Proteomics typically gives us a better understanding of an organism than genomics. First, the level of

transcription of a gene gives only a rough estimate of its level of expression into a

protein.[10]An mRNA produced in abundance may be degraded rapidly or translated inefficiently, resulting in a

small amount of protein. Second, as mentioned above many proteins experience post-translational

modifications that profoundly affect their activities; for example some proteins are not active until they become

phosphorylated. Methods such as phosphoproteomics andglycoproteomics are used to study post-translational

modifications. Third, many transcripts give rise to more than one protein, through alternative splicing or

alternative post-translational modifications. Fourth, many proteins form complexes with other proteins or RNA

molecules, and only function in the presence of these other molecules. Finally, protein degradation rate plays

an important role in protein content.[11]

Reproducibility. Proteomics experiments conducted in one laboratory are not easily reproduced in another. For

instance, Peng et al.[12] have identified 1504 yeast proteins in a proteomics experiment of which only 858 were

found in a similar previous study.[13] Further, the previous study identified 607 proteins that were not found by

Peng et al. This translates to a reproducibility of 57% (Peng vs. Washburn) to 59% (Washburn vs. Peng).

[edit]Methods of studying proteins

[edit]Identifying proteins that are post-translationally modified

One way in which a particular protein can be studied is to develop an antibody which is specific to that

modification. For example, there are antibodies which only recognize certain proteins when they are tyrosine-

phosphorylated, known as phospho-specific antibodies; also, there are antibodies specific to other

modifications. These can be used to determine the set of proteins that have undergone the modification of

interest.

For sugar modifications, such as glycosylation of proteins, certain lectins have been discovered which bind

sugars. These too can be used.[citation needed]

A more common way to determine post-translational modification of interest is to subject a complex mixture of

proteins to electrophoresis in "two-dimensions", which simply means that the proteins are electrophoresed first

in one direction, and then in another, which allows small differences in a protein to be visualized by separating

a modified protein from its unmodified form. This methodology is known as "two-dimensional gel

electrophoresis".[14]

Recently, another approach has been developed called PROTOMAP which combines SDS-PAGE with shotgun

proteomics to enable detection of changes in gel-migration such as those caused by proteolysis or post

translational modification.[15]

[edit]Determining the existence of proteins in complex mixtures

Classically, antibodies to particular proteins or to their modified forms have been used in biochemistry and cell

biology studies. These are among the most common tools used by practicing biologists today.

For more quantitative determinations of protein amounts, techniques such as ELISAs can be used.[citation needed]

For proteomic study, more recent techniques such as matrix-assisted laser desorption/ionization

(MALDI)[14] have been employed for rapid determination of proteins in particular mixtures and

increasingly electrospray ionization (ESI).[citation needed]

[edit]Computational methods in studying protein biomarkers

Computational predictive models[16] have shown that extensive and diverse feto-maternal protein trafficking

occurs during pregnancy and can be readily detected non-invasively in maternal whole blood. This

computational approach circumvented a major limitation, the abundance of maternal proteins interfering with

the detection of fetal proteins, to fetal proteomic analysis of maternal blood. Computational models can use

fetal gene transcripts previously identified in maternal whole blood to create a comprehensive proteomic

network of the term neonate. Such work shows that the fetal proteins detected in pregnant woman’s blood

originate from a diverse group of tissues and organs from the developing fetus. The proteomic networks

contain many biomarkers that are proxies for development and illustrate the potential clinical application of this

technology as a way to monitor normal and abnormal fetal development.

An information theoretic framework has also been introduced for biomarker discovery, integrating biofluid and

tissue information.[17] This new approach takes advantage of functional synergy between certain biofluids and

tissues with the potential for clinically significant findings not possible if tissues and biofluids were considered

individually. By conceptualizing tissue-biofluid as information channels, significant biofluid proxies can be

identified and then used for guided development of clinical diagnostics. Candidate biomarkers are then

predicted based on information transfer criteria across the tissue-biofluid channels. Significant biofluid-tissue

relationships can be used to prioritize clinical validation of biomarkers.[citation needed]

[edit]Establishing protein–protein interactions

Most proteins function in collaboration with other proteins, and one goal of proteomics is to identify which

proteins interact. This is especially useful in determining potential partners in cell signaling cascades.

Several methods are available to probe protein–protein interactions. The traditional method is yeast two-hybrid

analysis. New methods include protein microarrays, immunoaffinity chromatographyfollowed by mass

spectrometry, dual polarisation interferometry, Microscale Thermophoresis and experimental methods such as

phage display and computational methods

[edit]Practical applications of proteomics

One of the most promising developments to come from the study of human genes and proteins has been the

identification of potential new drugs for the treatment of disease. This relies on genome and proteome

information to identify proteins associated with a disease, which computer software can then use as targets for

new drugs. For example, if a certain protein is implicated in a disease, its 3D structure provides the information

to design drugs to interfere with the action of the protein. A molecule that fits the active site of an enzyme, but

cannot be released by the enzyme, will inactivate the enzyme. This is the basis of new drug-discovery tools,

which aim to find new drugs to inactivate proteins involved in disease. As genetic differences among individuals

are found, researchers expect to use these techniques to develop personalized drugs that are more effective

for the individual.[18]

[edit]Biomarkers

The FDA defines a biomarker as, "A characteristic that is objectively measured and evaluated as an indicator of

normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention".

Understanding the proteome, the structure and function of each protein and the complexities of protein–protein

interactions will be critical for developing the most effective diagnostic techniques and disease treatments in the

future.

An interesting use of proteomics is using specific protein biomarkers to diagnose disease. A number of

techniques allow to test for proteins produced during a particular disease, which helps to diagnose the disease

quickly. Techniques include western blot, immunohistochemical staining, enzyme linked immunosorbent

assay (ELISA) or mass spectrometry.[14][19] Secretomics, a subfield of proteomics that studies secreted

proteins and secretion pathways using proteomic approaches, has recently emerged as an important tool for

the discovery of biomarkers of disease.[20]

[edit]Proteogenomics

In what is now commonly referred to as proteogenomics, proteomic technologies such as mass

spectrometry are used for improving gene annotations. Parallel analysis of the genome and the proteome

facilitates discovery of post-translational modifications and proteolytic events,[21] especially when comparing

multiple species (comparative proteogenomics).[22]

[edit]Current research methodologies

Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE)[23] can be used to quantify variation in

the 2-D DIGE process and establish statistically valid thresholds for assigning quantitative changes between

samples.[24]

Comparative proteomic analysis can reveal the role of proteins in complex biological systems, including

reproduction. For example, treatment with the insecticide triazophos causes an increase in the content of

brown planthopper (Nilaparvata lugens (Stål)) male accessory gland proteins (Acps) that can be transferred to

females via mating, causing an increase in fecundity (i.e. birth rate) of females. [25] To identify changes in the

types of accessory gland proteins (Acps) and reproductive proteins that mated female planthoppers received

from male planthoppers, researchers conducted a comparative proteomic analysis of mated N.

lugens females.[26] The results indicated that these proteins participate in the reproductive process of N.

lugens adult females and males.[27]

Proteome analysis of Arabidopsis peroxisomes[28] has been established as the major unbiased approach for

identifying new peroxisomal proteins on a large scale.[29]

There are many approaches to characterizing the human proteome, which is estimated to contain between

20,000 and 25,000 non-redundant proteins. The number of unique protein species likely increase by between

50,000 and 500,000 due to RNA splicing and proteolysis events, and when post-translational modification are

also considered, the total number of unique human proteins is estimated to range in the low millions. [30][31]

In addition, first promising attempts to decipher the proteome of animal tumors have recently been reported. [14]

[edit]See also

Activity based proteomics

Bioinformatics

Bottom-up proteomics

Cytomics

Functional genomics

Genomics

Immunomics

Immunoproteomics

Lipidomics

List of biological databases

List of omics topics in biology

Metabolomics

PEGylation

Phosphoproteomics

Proteogenomics

Proteomic chemistry

Secretomics

Shotgun proteomics

Top-down proteomics

Systems biology

Transcriptomics

Yeast two-hybrid system

[edit]Protein databases

Cardiac Organellar Protein Atlas Knowledgebase (COPaKB)

Human Protein Reference Database

Model Organism Protein Expression Database (MOPED)

National Center for Biotechnology Information (NCBI)

Protein Data Bank (PDB)

Protein Information Resource (PIR)

Proteomics Identifications Database (PRIDE)

Proteopedia The collaborative, 3D encyclopedia of proteins and other molecules

Swiss-Prot

UniProt

[edit]Research centers

European Bioinformatics Institute

Netherlands Proteomics Centre (NPC)

Protein engineeringFrom Wikipedia, the free encyclopedia

Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with

much research taking place into the understanding of protein folding and recognition forprotein

design principles.

There are two general strategies for protein engineering, rational design and directed evolution. These

techniques are not mutually exclusive; researchers will often apply both. In the future, more detailed knowledge

of protein structure and function, as well as advancements in high-throughput technology, may greatly expand

the capabilities of protein engineering. Eventually, even unnatural amino acids may be incorporated thanks to

a new method that allows the inclusion of novel amino acids in the genetic code.

Contents

[hide]

1 Rational design of

proteins

2 Directed evolution

3 Examples of engineered

proteins

4 See also

5 References

6 External links

[edit]Rational design of proteins

Main article: Protein design

In rational protein design, the scientist uses detailed knowledge of the structure and function of the protein to

make desired changes. This generally has the advantage of being inexpensive and technically easy, since site-

directed mutagenesis techniques are well-developed. However, its major drawback is that detailed structural

knowledge of a protein is often unavailable, and even when it is available, it can be extremely difficult to predict

the effects of various mutations.

Computational protein design algorithms seek to identify novel amino acid sequences that are low in energy

when folded to the pre-specified target structure. While the sequence-conformation space that needs to be

searched is large, the most challenging requirement for computational protein design is a fast, yet accurate,

energy function that can distinguish optimal sequences from similar suboptimal ones.

[edit]Directed evolution

Main article: Directed evolution

In directed evolution, random mutagenesis is applied to a protein, and a selection regime is used to pick out

variants that have the desired qualities. Further rounds of mutation and selection are then applied. This method

mimics natural evolution and generally produces superior results to rational design.An additional technique

known as DNA shuffling mixes and matches pieces of successful variants in order to produce better results.

This process mimics the recombination that occurs naturally during sexual reproduction. The great advantage

of directed evolution is that it requires no prior structural knowledge of a protein, nor is it necessary to be able

to predict what effect a given mutation will have. Indeed, the results of directed evolution experiments are often

surprising in that desired changes are often caused by mutations that were not expected to have that effect.

The drawback is that they require high-throughput, which is not feasible for all proteins. Large amounts

of recombinant DNA must be mutated and the products screened for desired qualities. The sheer number of

variants often requires expensive robotic equipment to automate the process. Furthermore, not all desired

activities can be easily screened for.

[edit]Examples of engineered proteins

Using computational methods, a protein with a novel fold has been designed, known as Top7,[1] as well as

sensors for unnatural molecules.[2] The engineering of fusion proteins has yieldedrilonacept, a pharmaceutical

which has secured FDA approval for the treatment of cryopyrin-associated periodic syndrome.

Another computational method, IPRO, successfully engineered the switching of cofactor specificity of Candida

boidinii xylose reductase.[3] Iterative Protein Redesign and Optimization (IPRO) redesigns proteins to increase

or give specificity to native or novel substrates and cofactors. This is done by repeatedly randomly perturbing

the backbones of the proteins around specified design positions, identifying the lowest energy combination of

rotamers, and determining if the new design has a lower binding energy than previous ones. The iterative

nature of this process allows IPRO to make additive mutations to the protein sequence that collectively improve

the specificity towards the desired substrates and/or cofactors. Details on how to download the software

implemented in Python and experimental testing of predictions are outlined in the following paper. [4]

Computation-aided design has also been used to engineer complex properties of a highly ordered nano-protein

assembly. [5] A protein cage, E. coli bacterioferritin (EcBfr), which naturally shows structural instability and an

incomplete self-assembly behavior by populating two oligomerization states is the model protein in this study.

Through computational analysis and comparison to its homologs, it has been found that this protein has a

smaller than average dimeric interface on its two-fold symmetry axis mainly due to the existence of an

interfacial water pocket centered around two water-bridged asparagine residues. To investigate the possibility

of engineering EcBfr for modified structural stability, a semi-empirical computational method is used to virtually

explore the energy differences of the 480 possible mutants at the dimeric interface relative to the wild-type

EcBfr. This computational study also converges on the water-bridged asparagines. Replacing these two

asparagines with hydrophobic amino acids results in proteins that fold into alpha-helical monomers and

assemble into cages as evidenced by circular dichroism and transmission electron microscopy. Both thermal

and chemical denaturation confirm that, all redesigned proteins, in agreement with the calculations, possesses

increased stability. One of the three mutations shifts the population in favor of the higher order oligomerization

state in solution as shown by both size exclusion chromatography and native gel electrophoresis. [5]

[edit]See also

Display:

Bacterial display

Phage display

mRNA display

Ribosome display

Yeast display

Biomolecular engineering

Enzyme engineering

Enzymology

Expanded genetic code

Gene synthesis

Meganucleases

Nucleic acid analogues

Protein folding

Protein design

Proteomics

Proteome

SCOPE (protein engineering)

Structural biology

Synthetic biology

Immobilized enzyme

Immobilized enzymeFrom Wikipedia, the free encyclopedia

An immobilized enzyme is an enzyme that is attached to an inert, insoluble material such as calcium alginate

(produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride). This

can provide increased resistance to changes in conditions such as pH or temperature. It also allows enzymes

to be held in place throughout the reaction, following which they are easily separated from the products and

may be used again - a far more efficient process and so is widely used in industry

for enzyme catalysed reactions. An alternative to enzyme immobilization is whole cell immobilization.

[edit]Commercial use

Immobilized enzymes are very important for commercial uses as they possess many benefits to the expenses

and processes of the reaction of which include:

Convenience: Minuscule amounts of protein dissolve in the reaction, so workup can be much easier.

Upon completion, reaction mixtures typically contain only solvent and reaction products.

Economical: The immobilized enzyme is easily removed from the reaction making it easy to recycle

the biocatalyst.

Stability: Immobilized enzymes typically have greater thermal and operational stability than the

soluble form of the enzyme.

In the past, biological washing powders and detergents would contain many proteases and lipases which would

break down dirt. However, when the cleaning products would come into contact with the skin, it would create

allergic reactions. This is why immobilization of enzymes are important, not just economically.

[edit]Immobilization of an Enzyme

There are three different ways by which one can immobilise an enzyme, which are the following, listed in order

of effectiveness:

Adsorption on glass, alginate beads or matrix: Enzyme is attached to the outside of an inert

material. In general, this method is the slowest among those listed here. As adsorption is not a chemical

reaction, the active site of the immobilized enzyme may be blocked by the matrix or bead, greatly reducing

the activity of the enzyme.

Entrapment: The enzyme is trapped in insoluble beads or microspheres, such as calcium

alginate beads. However, this insoluble substances hinders the arrival of the substrate, and the exit of

products.

Cross-linkage: The enzyme is covalently bonded to a matrix through a chemical reaction. This

method is by far the most effective method among those listed here. As the chemical reaction ensures that

the binding site does not cover the enzyme's active site, the activity of the enzyme is only affected by

immobility. However, the inflexibility of the covalent bonds precludes the self-healing properties exhibited

by chemoadsorbed self-assembled monolayers. Use of a spacer molecule like poly(ethylene glycol) helps

reduce the steric hindrance by the substrate in this case. Enzymes may also be immobilized to a surface

using non-covalent or covalent Protein tags.

[edit]External links

Kinetic Perfection

Kinetic perfectionFrom Wikipedia, the free encyclopedia

(Redirected from Kinetic Perfection)

Kinetic perfection, also known as catalytic perfection, refers to enzymes that are diffusion-limited; that is,

the reaction they catalyze occurs as quickly as the reactants diffuse to the enzyme. The time needed for the

reaction to occur is negligible compared to the time it takes for the substrate to enter, and product to leave,

the active site.

Kinetic perfection: "Their [the enzymes] catalytic velocity is restricted only by the rate at which they [the

enzymes] encounter substrates in a solution."[1]

This means that the rate of the enzyme catalysed reaction is actually limited by diffusion. The enzyme

'processes' the substrate well before it encounters another molecule. An example of a kinetically perfect

enzyme is Triose phosphate isomerase, which is involved in the glycolytic pathway.

Some kinetically perfect enzymes may employ the 'Circe Effect', the use of electostatic forces to attract

substrate molecules into active sites.

It is worth noting that there are not many kinetically perfect enzymes. This can be explained in terms of natural

selection. An increase in catalytic speed may be favoured as it could confer some advantage to the organism.

However, when the catalytic speed outstrips diffusion speed (i.e. substrates entering and leaving the active

site, and also encountering susbstrates) there is no more advantage to increase the speed even further.

Increasing the catalytic speed past the diffusion speed will not aid the organism in any way. Therefore these

perfect enzymes must have come about by 'lucky' random mutation which happened to spread, or because the

faster speed was once useful as part of a different reaction in the enzyme's ancestry.

Enzyme engineeringFrom Wikipedia, the free encyclopedia

Enzyme engineering (or enzyme technology) is the application of modifying an enzyme's structure (and thus

its function) or modifying the catalytic activity of isolated enzymes to produce new metabolites, to allow new

(catalyzed) pathways for reactions to occur,[1] or to convert from some certain compounds into others

(biotransformation). These products will be useful as chemicals, pharmaceuticals, fuel, food or agricultural

additives.

An enzyme reactor [2] consists of a vessel containing a reactional medium that is used to perform a desired

conversion by enzymatic means. Enzymes used in this process are free in the solution.

Enzyme engineering

EnzymesEnzymes are catalysts. Mostare proteins. (Afew ribonucleoprotein enzymes have been discovered and,for some of these, the catalyticactivity is in the RNA partrather than the protein part.Link to discussion ofthese ribozymes.)

Enzymes bind temporarily to one or more of the reactants — the substrate(s) — ofthe reaction they catalyze. In doing so, they lower the amount of activationenergyneeded and thus speed up the reaction.

Examples:

Index to this page

Enzymes Competitive inhibition Enzyme cofactors Lysozyme: a model of enzyme action Factors Affecting Enzyme Action Regulation of Enzyme Activity

o Anchoring enzymes in membraneso Inactive precursorso Feedback Inhibitiono Precursor Activation

Regulation of Enzyme Synthesis

Link to a discussion of free energy (G) and "ΔG".

Catalase. It catalyzes the decomposition of hydrogen peroxide into water andoxygen.

2H2O2 -> 2H2O + O2

One molecule of catalase can break 40 million molecules of hydrogen peroxideeach second.

Carbonic anhydrase. It is found in red blood cells where it catalyzes thereaction

CO2 + H2O ↔ H+ + HCO3−

It enables red blood cells to transport carbon dioxide from the tissues to thelungs. [Discussion]

One molecule of carbonic anhydrase can process one million molecules ofCO2 each second.

Acetylcholinesterase. It catalyzes the breakdown ofthe neurotransmitter acetylcholine at several types of synapses as well as atthe neuromuscular junction — the specialized synapse that triggers thecontraction of skeletal muscle.

One molecule of acetylcholinesterase breaks down 25,000 molecules ofacetylcholine each second. This speed makes possible the rapid "resetting" ofthe synapse for transmission of another nerve impulse.

Enzyme activity can be analyzed quantitatively. Some of the ways this is done are described inthe page Enzyme Kinetics. Link to it.

In order to do its work, an enzyme must unite — even if ever so briefly — with atleast one of the reactants. In most cases, the forces that hold the substrate in the activesite of the enzyme are noncovalent, an assortment of:

hydrogen bonds ionic interactions and hydrophobic interactions

Link to discussion of the noncovalent forces that hold macromolecules together.

Most of these interactions are weak andespecially so if the atoms involved are fartherthan about one angstrom from each other. Sosuccessful binding of the substrate in theactive site of the enzyme requires that the twomolecules be able to approach each otherclosely over a fairly broad surface. Thus theanalogy that a substrate molecule binds its enzyme like a key in a lock.

This requirement for complementarity in the configuration of substrate and enzymeexplains the remarkable specificity of most enzymes. Generally, a given enzyme isable to catalyze only a single chemical reaction or, at most, a few reactions involvingsubstrates sharing the same general structure.

Competitive inhibition

The necessity for aclose, if brief, fitbetween enzyme andsubstrate explains thephenomenon ofcompetitiveinhibition.

One of the enzymes needed for the release of energy within the cell is succinicdehydrogenase.Link to illustrateddiscussion of thecitric acid cycle.

It catalyzes the oxidation (by the removal of two hydrogen atoms) of succinic acid (a).If one adds malonic acid to cells, or to a test tube mixture of succinic acid and theenzyme, the action of the enzyme is strongly inhibited. This is because the structure ofmalonic acid allows it to bind to the same site on the enzyme (b). But there is nooxidation so no speedy release of products. The inhibition is called competitivebecause if you increase the ratio of succinic to malonic acid in the mixture, you willgradually restore the rate of catalysis. At a 50:1 ratio, the two molecules compete onroughly equal terms for the binding (=catalytic) site on the enzyme.Link to a quantitative treatment of competitive inhibition.

Enzyme cofactors

Many enzymes require the presence of an additional, nonprotein, cofactor.

Some of these are metal ions such as Zn2+ (the cofactor for carbonicanhydrase), Cu2+, Mn2+, K+, and Na+.

Some cofactors are small organic molecules called coenzymes. The B vitaminso thiamine (B1)o riboflavin (B2) ando nicotinamide

are precursors of coenzymes.

Coenzymes may be covalently bound to the protein part (called the apoenzyme) ofenzymes as a prosthetic group. Others bind more loosely and, in fact, may bind onlytransiently to the enzyme as it performs its catalytic act.

Lysozyme: a model ofenzyme action

A number of lysozymes arefound in nature; in human tearsand egg white, for examples.The enzyme is antibacterialbecause it degrades thepolysaccharide that is found inthe cell walls of many bacteria.

It does this by catalyzing the insertion of a water molecule at the position indicated bythe red arrow (a glycosidic bond). This hydrolysis breaks the chain at that point.Link to discussion of the bacterialcell wall and how it is affectedby certain antibiotics.

Link to view of the primary structure ofhen's egg white lysozyme.

The bacterial polysaccharide consists of long chains of alternating amino sugars:

N-acetylglucosamine (NAG) N-acetylmuramic acid (NAM)

These hexose units resemble glucose except for the presence of the side chainscontaining amino groups.

Lysozyme is a globular protein with a deep cleftacross part of its surface. Six hexoses of the substratefit into this cleft.

With so many oxygen atoms in sugars, as manyas 14 hydrogen bonds form between the sixamino sugars and certain amino acid Rgroups such as Arg-114, Asn-37, Asn-44,Trp-62, Trp-63, and Asp-101.

Some hydrogen bonds also form with the C=Ogroups of several peptide bonds.

In addition, hydrophobic interactions may helphold the substrate in position.

X-ray crystallography has shown that as lysozyme andits substrate unite, each is slightly deformed. Thefourth hexose in the chain (ring #4) becomes twistedout of its normal position. This imposes a strain on theC-O bond on the ring-4 side of the oxygen bridgebetween rings 4 and 5. It is just at this point that thepolysaccharide is broken. A molecule of water isinserted between these two hexoses, which breaks thechain. Here, then, is a structural view of what it meansto lower activation energy. The energy needed to break this covalent bond is lowernow that the atoms connected by the bond have been distorted from their normalposition.

As for lysozyme itself, binding of the substrate induces a small (~0.75Å) movementof certain amino acid residues so the cleft closes slightly over its substrate. So the"lock" as well as the "key" changes shape as the two are brought together. (This issometimes called "induced fit".)

The amino acid residues in the vicinity of rings 4 and 5 provide a plausiblemechanism for completing the catalytic act. Residue 35, glutamic acid (Glu-35), isabout 3Å from the -O- bridge that is to be broken. The free carboxyl group ofglutamic acid is a hydrogen ion donor and available to transfer H+ to the oxygen atom.This would break the already-strained bond between the oxygen atom and the carbonatom of ring 4.

Now having lost an electron, the carbon atom acquires a positive charge. Ionizedcarbon is normally very unstable, but the attraction of the negatively-charged carboxylion of Asp-52could stabilize it long enough for an -OH ion (from a spontaneouslydissociated water molecule) to unite with the carbon. Even at pH 7, waterspontaneously dissociates to produce H+and OH- ions. [Discussion] The hydrogen ion(H+) left over can replace that lost by Glu-35.

In the 20 August 2001 issue of Nature, Vocadlo, D. J., et al., report evidence that Asp-52stabilizes ring 4 by forming a transient covalent bond rather than through ionic interactions.

In either case, the chain is broken, the two fragments separate from the enzyme, andthe enzyme is free to attach to a new location on the bacterial cell wall and continueits work of digesting it.

Factors Affecting Enzyme Action

The activity of enzymes is strongly affected by changes in pH and temperature. Eachenzyme works best at a certain pH (left graph) and temperature (right graph), itsactivity decreasing at values above and below that point. This is not surprisingconsidering the importance of

tertiary structure (i.e. shape) in enzymefunction and

noncovalent forces, e.g., ionic interactionsand hydrogen bonds, in determining thatshape.

Examples:

the protease pepsin works best as a pH of 1–2 (found in the stomach) while the protease trypsin is inactive at such a low pH but very active at a pH of 8

(found in the small intestine as the bicarbonate of the pancreatic fluidneutralizes the arriving stomach contents). [Discussion]

Changes in pH alter the state of ionization of charged amino acids (e.g., Asp, Lys) thatmay play a crucial role in substrate binding and/or the catalytic action itself. Withoutthe unionized -COOH group of Glu-35 and the ionized -COO- of Asp-52, the catalyticaction of lysozyme would cease.

Hydrogenbonds are easilydisrupted byincreasingtemperature.This, in turn,may disrupt theshape of theenzyme so thatits affinity for itssubstrate

diminishes. The ascending portion of the temperature curve (red arrow in right-handgraph above) reflects the general effect of increasing temperature on the rate ofchemical reactions (graph at left). The descending portion of the curve above (bluearrow) reflects the loss of catalytic activity as the enzyme moleculesbecome denatured at high temperatures.

Regulation of Enzyme Activity

Several mechanisms work to make enzyme activity within the cell efficient and well-coordinated.

Anchoring enzymes in membranes

Many enzymes are inserted into cell membranes, for examples,

the plasma membrane the membranes of mitochondria and chloroplasts the endoplasmic reticulum the nuclear envelope

These are locked into spatial relationships that enable them to interact efficiently.

Inactive precursors

Enzymes, such as proteases, that can attack the cell itself are inhibited while withinthe cell that synthesizes them. For example, pepsin is synthesized within the chiefcells (in gastric glands) as an inactive precursor,pepsinogen. Only when exposed to

Distribution of energies in twopopulations of molecules, onemaintained at a low temperature,the other at a higher temperature.The arrow represents the thresholdenergy needed for these moleculesto react chemically. At highertemperatures, a larger proportion ofthe molecules exceed the thresholdenergy, and the reaction proceedsmore rapidly.

the low pH outside the cell is theinhibiting portion of the moleculeremoved and active pepsinproduced.

Feedback Inhibition

If the product of a series ofenzymatic reactions, e.g., an amino acid, begins to accumulate within the cell, it mayspecifically inhibit the action of the first enzyme involved in its synthesis (red bar).Thus further production of the enzyme is halted.

Precursor Activation

The accumulation of a substance within a cell may specifically activate (blue arrow)an enzyme that sets in motion a sequence of reactions for which that substance is theinitial substrate. This reduces the concentrationof the initial substrate.

In the case if feedback inhibition and precursoractivation, the activity of the enzyme is beingregulated by a molecule which is not itssubstrate. In these cases, the regulator moleculebinds to the enzyme at a different site than theone to which the substrate binds. When theregulator binds to its site, it alters the shape of the enzyme so that its activity ischanged. This is called an allosteric effect.

In feedback inhibition, the allosteric effect lowers the affinity of the enzyme forits substrate.

In precursor activation, the regulator molecule increases the affinity of theenzyme in the series for its substrate.

Regulation of Enzyme Synthesis

The four mechanisms described above regulate the activity of enzymes alreadypresent within the cell.

What about enzymes that are not needed or are needed but not present?

Here, too, control mechanisms are at work that regulate the rate at which newenzymes are synthesized. Most of these controls work by turning on — or off —the transcription of genes.

If, for example, ample quantities of an amino acid are already available to the cellfrom its extracellular fluid, synthesis of the enzymes that would enable the cell toproduce that amino acid for itself is shut down.

Conversely, if a new substrate is made available to the cell, it may induce thesynthesis of the enzymes needed to cope with it. Yeast cells, for example, do notordinarily metabolize lactose, and no lactase can be detected in them. However, ifgrown in a medium containing lactose, they soon begin synthesizing lactase — bytranscribing and translating the necessary gene(s) — and so can begin to metabolizethe sugar.

Link to a discussion of how transcription of a gene needed for lactose metabolism is controlled inE. coli.

E. coli also has a mechanism which regulates enzyme synthesis bycontrolling translation of a needed messenger RNA. Link to a discussion.

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14 April 2011