ENZYMES• They control metabolism by regulating metabolic reaction rates:

molecules that accelerate or catalyze chemical reactions in cells by breaking old covalent bonds & forming new covalent bonds                                                 

• Except for Ribozymes, all enzymes are proteins

• a biological catalyst…• have complex structure (sequence of aa’s) • act only upon a specific substrate (or substrate group)• do not change the energetics of the reaction

ENZYME ACTİON  E + S <---> [ES] <---> E + P

enzymes catalyze reactions by lowering the energy of activation (Ea)                    

  WHAT DOES AN ES COMPLEX DO?- holds substrate out of aqueous solution- holds substrate in specific orientation, close to Transition

State to allow reaction to occur- reduces ability of free rotation & molecular collisions with

non-reactive atoms- allows an altered local environment: changes ionic strength,

pH, adds or removes H-bonds to substrate

TERMİNOLOGYMany enzymes require a non-protein component for activity:

cofactor: small inorganic ions... mostly metal ions: Cu (cytochrome oxidase), Mg (kinases), Fe (catalase, peroxidase) 

coenzymes: small non-protein but organic compounds   Coenzyme A: acyl transfer

Flavins: redox reactionNAD+ (NADP+): redox reactions Vitamins: derivatives of B vitamins (B1, B2, B6, B12), niacin, folic acid, riboflavin

prosthetic group: tightly bound large complex organic molecules, (heme)

Holoenzyme vs apoenzyme (apoprotein)

• active site: portion of enzyme which folds to precisely fit the contours of a substrate via weak electrostatic interactions & facilitates bond reactivity

• allosteric site: a site other than the active site

Isoenzymes

Classification is based on reaction catalyzed so enzymes isolated from different organisms but catalysing same rxn have same number but different amino acid sequence

Even within a single species, there may exist different forms of enzyme catalysing the same reaction. Differences may be: A.acid sequence Some covalent modification 3-D structure

Isoenzyme (isozyme): different variants of the same enzyme having identical functions

PROPERTİES OF ENZYMES AS CATALYSTS-1Catalytic power They may increase reaction rate by as much as 1015-fold

2H2O2 2H2O + O2 Rate (L/mol/s)No catalyst 1 x 10-7

Fe2+ catalyst 56Catalase 4 x 107

Specificity Most enzymes are highly specific to their substrate and

reaction catalysed Bond specificity: e.g peptidase, phosphatase Group specificity: e.g hexokinase Absolute or near-absolute specificity

Stereospecificity: Dehydrogenases catalyst the transfer of hydrogen from the

substrate to a particular side of nicotinamide ring in NAD+ or NADP+

Phenylalanine hydroxylase uses L-Phe not D-Phe Importance of specificity in DNA replication and protein

synthesis proofreading

PROPERTİES OF ENZYMES AS CATALYSTS-2Regulation Allosteric regulation (+/- effectors)

e.g. feedback inhibition Covalent regulation (phosphorylation by ATP-dependent

protein kinases) e.g. Glycogen phosphorylase

Activation of zymogens, which are inactive proenzymese.g. trypsinogen

Amount of enzyme: gene expression enzyme degradation

HOW TO DEFİNE ENZYME ACTİVİTY?

Physical properties of an enzyme most often is measured by relative rate that  substrate ---> product            

1 unit ACTIVITY= International unit (IU)amount enzyme which converts 1 μmole substrate per

min at 25oC e.g. IU= 10 μmole/min

1 unit SPECIFIC ACTIVITY# IU of enzymatic activity per mg of total protein present e.g. 10 μmole/min/mg protein or 10 IU/mg protein

  

CLASSİFİCATİON OF ENZYMES

Enzyme Commission (EC, 1955) - IUBMB International Union of Biochemistry & Molecular Biology                                         

  4 digit Numbering System    [1.2.3.4]

1st one of the 6 major classes of enzyme activity2nd the subclass (type of substrate or bond cleaved)3rd the sub-subclass (group acted upon, cofactor required,

etc...)4th a serial number… (order in which enzyme was added to

list)

MAJOR CLASSES OF ENZYMES-11. Oxidoreductases [dehydrogenases, oxidases, peroxidases]

oxidation-reduction reactions, often using coenzyme as NAD+/FAD

Alcohol dehydrogenase [EC 1.1.1.1]  CH3CH2OH + NAD+ ---> CH3CHO + NADH + H+  

2. Transferases [kinase, phosphorylase, transaminases] group transfer reactions (AX + B BX + A)

Hexokinase   [EC 2.7.1.2]   D-glu + ATP ---> D-glu-6-P + ADP   

3. Hydrolases [digestive enzymes; amylases, lactase, sucrase] hydrolytic reactions: (AX + H2O XOH +

HA) Alkaline phosphatase   [EC 3.1.3.1]   R-PO4 + H2O ---> R-OH + H-PO4  

 

MAJOR CLASSES OF ENZYMES-24. Lyases [decarboxylases] elimination rxns in which a

double bond is formed     Pyruvate decarboxylase  [EC 4.1.1.1]    pyruvate ---> acetaldehyde + CO2    

5. Isomerases [mutases, cis-trans isomerases, racemases]  isomerization rxns                              

Alanine racemase  [EC 5.1.1.1]  L-alanine ---> D-alanine  

6. Ligases [a.acid RNA ligase] condensation of 2 substrates at the expense of energy (ATP)(X + Y + ATP XY + ADP + Pi)

Isoleucine-tRNA ligase [EC 6.1.1.5]   L-isoleucine + tRNAIle + ATP ---> L-isoleucyl- tRNAIle + ADP + PPi

MULTİENZYME SYSTEMS Proteins that exhibit more than one catalytic activity EC recommendation more than one catalytic activity

systeme.g. fatty acid synthase system

Multifunctional enzymes will have more than one EC number...

Multifunctional enzyme can made up of: Several polypeptide chains with different catalytic

activities may be associated with each other A single polypeptide chain with multiple catalytic site or even both

Enzymes are used in industrial processes and as analytical reagents in medicine

Immobilisation of enzymes is an important technique used in industry as it enables economical operation of a process and protection of

enzymes during their use

Because of their sensitivity and specificity, enzymes are used as analytical reagents in systems such as the detection of glucose in

human blood and urine

Thermostability and an ability to withstand extremes of pH are

essential properties for enzymes usedin many industrial processes

Enzymes in Biotechnology

Enzyme technology is concerned with the application of enzymesas tools of industry, agriculture and medicine

Enzymes are biological catalysts that fulfil their roleby binding specific substrates at their active sites

This specificity is one property of enzymes thatmakes them useful for industrial applications

The value of using enzymes over inorganic catalysts in the technological field is their efficiency, selectivity and specificity

Enzymes are able to operate at room temperature, atmospheric pressure and within normal pH ranges (around 7)– all of which create energy savings for industry

Enzymes possess specifically shaped active sites for reacting with one specific substrate thereby generating pure products

free from unwanted by-products

Enzymes are biodegradable and, unlike many inorganiccatalysts, cause less damage to the environment

Enzyme Technology

The micro-organisms(such as yeast) are really

used as a source of enzymes during the manufacture of

these products of biotechnology

Many industrial processes now make use of pure sources of enzymes, i.e. the enzymes have been ISOLATED from the micro-organisms before use

Micro-organisms have beenused for thousands of yearsfor making products such as

wine, beer, vinegar, soy sauce,bread and cheese

Products of Enzyme Technology

The large scale production of enzymes involves culturing micro-organismsin chambers called FERMENTERS or BIOREACTORS

Micro-organisms are suitable for use in the large scale production of enzymes in fermenters because:

• They have rapid growth rates and are able to produce larger numbers of enzyme molecules per body mass than many other organisms

• Micro-organisms can be genetically engineered to improve the strain and enhance yields

• Micro-organisms are found in a wide variety of different habitats such that their enzymes are able to function across a range of temperatures and pH

• Micro-organisms have simple growth requirements and these can be precisely controlled within the fermenter

• Micro-organisms can utilise waste products such as agricultural waste as substrates

Large Scale Production of Enzymes

The costs associated with the use of enzymes for industrial purposes can also be reduced by immobilising the enzymes

Enzymes for industrial processes are more valuable when they are able to act in an insolubilised state rather than in solution

Enzymes are immobilised by binding them to, or trapping them in a solid support

Various methods for immobilising enzymes are available

Immobilised Enzymes

Enzymes are held on to a solidsupport (matrix) by weak forcessuch as hydrogen bonding

Enzymes are trapped withinthe structure of a solid polymer(usually in the form of beads)– the enzyme is trapped ratherthan bound

Methods for Immobilising Enzymes

Enzymes are covalently bondedto a matrix such as celluloseor collagen

Another more expensive method involvesenzymes which are both covalently bondedto, and cross-linked within, a matrix

Cross-linking and covalent bonding maycause some enzymes to lose their catalyticactivity especially if the active site is involvedin forming the linkages

Compared with free enzymes in solution, immobilised enzymeshave a number of advantages for use in industrial processes

The stability of many enzymes is increased when they are in an immobilised state; they are less susceptible to changes in

environmental conditions such as temperature and pH fluctuations

Immobilised enzymes can be recovered and re-used,reducing overall costs

The products of the reaction are not contaminated with enzyme eliminating the need to undertake costly separation of

the enzyme from the product

Immobilising enzymes allows for continuous production of a substance with greater automation

Advantages of Immobilising Enzymes

Enzyme Immobilisation and Thermostable Enzymes inThe Production of High Fructose Syrup

This industrial process involves the conversion of cheap corn starch into a high fructose syrup for use as a sweetener in confectionary and drinks

Starch Paste Starch paste is incubated with thethermostable enzyme alpha amylase

at 90oC for a couple of hours

Dextrins(short chains

of glucosemolecules)

Alpha amylase catalyses the hydrolysis of the starchinto short glucose chains called dextrins

The temperature is raised to 140oC to denature theamylase and then lowered to around 55oC before

adding the fungal enzyme amyloglucosidase

Glucose

Amyloglucosidase catalyses the hydrolysis ofdextrins into glucose molecules

Fructose syrup emergesfrom the end of the column

free from contaminationwith enzyme

The final stage involvesthe conversion of glucose

syrup into the much sweeterfructose syrup using the

enzyme glucose isomerase

Glucose isomerase is immobilisedin rigid granules and packed into

a column

Glucose syrup is poured intothe top of the column and ishydrolysed as it contacts the

immobilised enzyme

The sensitivity and specificity of enzymes makes them usefultools in medicine for the detection and measurement of chemicals

in fluids such as blood and urine

Because of their specificity, enzymes will bind to only one substrate – they can therefore be used for the identification

of a specific substance in a biological sample

Because of their sensitivity, enzymes are able to detect thepresence of specific molecules even when they are

present at very low concentrations

The enzyme glucose oxidase is used in an immobilised formfor the detection of glucose in biological fluids

Enzymes as Analytical Agents

The colour of the pad on the clinistix is compared witha colour chart to determine the amount of glucose

present in the sample

Increasing amounts of glucoseNoglucose

Glucose Measurement using 'Clinistix'