notes on enzyme

16/07/08 Enzyme Pharmaceutical

DHN CHANDAN – Department of ACCT, RU (Mobile – 88-01713413431) 1

1. What is enzyme? Enzyme: Enzyme may be defined as a soluble colloidal biocatalyst which is produced by living cells,

protein in nature (exception – RNA acting as ribozyme), specific in action, capable of catalyzing a chemical reaction without being altered or destroyed at the end of the process and does not change the equilibrium constant of the reaction but increases the rate at which the reaction approaches equilibrium. Or,

Enzyme may be defined as a reaction specific, thermo-labile, non-dialyzable protein catalyst, produced by the living cells, capable of catalyzing a bio-chemical reaction and reverts to its original state when the reaction is over.

Most of the enzymes are soluble in water. These are inactive at 0℃ and destroyed by heating at 100℃. Enzymes promote and control the conversion of the complex carbohydrates, proteins, fats into simple substances which intestine can absorb. They are usually colorless but some are yellow, blue, green etc. the very existence of life is unimaginable without the presence of enzymes.

2. What is the nature of enzyme? On which factors stability of enzyme depends? Nature: Enzymes are protein in nature. So they can be inactivated or destroyed by excessive heat

and are precipitated by salts of heavy metals. Factors: Following are the factors on which enzyme activity depends:

i. 𝑝𝐻 ii. Temperature and

iii. Concentration of a solution.

3. Why enzymes are not dialyzable? Enzymes are made of protein. As proteins cannot be dialyzed, so enzymes are not dialyzable.

4. Tell the division of enzyme. Chemically enzymes are divided into two groups:

a) Simple protein enzyme: They consist of simple protein. b) Complex protein enzyme or conjugate enzyme: They consist of a protein part, apoenzyme

and a prosthetic group, coenzyme. Combination of apoenzyme with coenzyme constitutes the holoenzyme. Thus holoenzyme is conjugate enzyme.

5. How can you classify enzymes? Principally enzymes are three classes. They are:

A. Coenzyme: These are the enzymes that required some non-protein compounds known as co-factor in order to perform their catalytic activity.

B. Apoenzyme: When enzymes do not possess the co-factor, the remaining conjugated proteins are called apoenzymes.

C. Holoenzyme: When enzymes possess chemical groups that are non-amino acid in nature, these conjugated proteins are called holoenzymes.

Enzymes are sometimes considered under two broad categories. They are:

A. Intracellular enzymes: They are functional within cells where they are synthesized. B. Extracellular enzymes: These enzymes are active outside the cell (e.g. digestive enzymes).


2 DHN CHANDAN – Department of ACCT, RU (Mobile – 88-01713413431)

The International Commission of Enzyme in 1961 has recommended a systematic method of nomenclature and classification of enzymes. According to this system enzymes are divided into six main groups depending upon their chemical reaction type and reaction mechanism. They are:

A. Oxidoreductases: Reaction catalyzed: Catalyze oxidation-reduction reaction between two substrates. The

hydrogen donor regarded as the substrate. 𝐴𝐻2 + 𝐵 → 𝐴 + 𝐵𝐻2

Examples: Alcohol dehydrogenase, cytochrome oxidase, L-amino acid oxidase, R-amino acid oxidase, lactate dehydrogenase etc.

Specific reactions: i. Lactate dehydrogenase oxidizes lactic acid to pyruvic acid. This enzyme is anaerobic

dehydrogenase as it uses some other substance as hydrogen acceptor. 𝐶𝐻3 − 𝐶𝐻 − 𝐶𝑂𝑂

−

| 𝑂𝐻

+ 𝑁𝐴𝐷+𝒍𝒂𝒄𝒕𝒂𝒕𝒆 𝒅𝒆𝒉𝒚𝒅𝒓𝒐𝒈𝒆𝒏𝒂𝒔𝒆→

𝐶𝐻3 − 𝐶 − 𝐶𝑂𝑂−

|| 𝑂

+ 𝑁𝐴𝐷𝐻 + 𝐻+

Lactate ion Pyruvate ion ii. Alcohol dehydrogenase oxidizes alcohol to aldehyde or ketone.

Alcohol + 𝑁𝐴𝐷+𝒂𝒍𝒄𝒐𝒉𝒐𝒍 𝒅𝒆𝒉𝒚𝒅𝒓𝒐𝒈𝒆𝒏𝒂𝒔𝒆→ Aldehyde / Ketone + 𝑁𝐴𝐷𝐻 +𝐻+

B. Transferases: Reaction catalyzed: Catalyze the transfer of some group or radical containing 𝐶,𝑁 and 𝑃

(other than hydrogen). 𝐴-𝑋 + 𝐵 → 𝐴 + 𝐵-𝑋

Examples: Hexokinase, transaminases, transmethylases, phosphorylase, choline acyl transferase etc.

Specific reactions: i. Aminotransferase catalyzes the transfer of nitrogen containing group.

𝐶𝑂𝑂− | 𝐶 = 𝑂| 𝐶𝐻2 | 𝐶𝐻2 | 𝐶𝑂𝑂−

+

𝐶𝑂𝑂−

|

𝐻3𝑁+ − 𝐶 − 𝐻 | 𝑅

𝒂𝒎𝒊𝒏𝒐𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒂𝒔𝒆→

𝐶𝑂𝑂− |

𝐻3𝑁+ − 𝐶 − 𝐻

| 𝐶𝐻2 | 𝐶𝐻2 | 𝐶𝑂𝑂−

+

𝐶𝑂𝑂−

| 𝐶 = 𝑂| 𝑅

α-ketoglutaric acid glutamate α-keto acid ii. Serine hydroxyl-methyl transferase catalyzes the transfer of carbon. 𝐶𝐻2 − 𝐶𝐻 − 𝐶𝑂𝑂

−

| |

𝑂𝐻 𝑁𝐻3+

+ 𝑇𝐻𝐹𝒔𝒆𝒓𝒊𝒏𝒆 𝒉𝒚𝒅𝒓𝒐𝒙𝒚−𝒎𝒆𝒕𝒉𝒚𝒍 𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒂𝒔𝒆→

𝐶𝐻2 − 𝐶𝑂𝑂−

|

𝑁𝐻3+

+𝑇𝐻𝐹\ / 𝐶𝐻2

Serine Glysine iii. Choline acyl transferase catalyzes the transfer of acyl-containing group.

acetyl-𝐶𝑜𝐴 + choline𝒄𝒉𝒐𝒍𝒊𝒏𝒆 𝒂𝒄𝒆𝒕𝒚𝒍 𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒂𝒔𝒆→ 𝐶𝑜𝐴 + 𝑂-acetyl choline

iv. Hexokinase catalyzes transfer of phosphorous containing group.

𝐴𝑇𝑃 + 𝐷-hexose𝒉𝒆𝒙𝒐𝒌𝒊𝒏𝒂𝒔𝒆→ 𝐴𝐷𝑃 + 𝐷-hexose-6-phosphate

C. Hydrolases: Reaction catalyzed: Catalyze cleavage of bonds (e.g. ester, ether, peptide, glycosyl, acid

anhydride etc.) by addition of water.



𝐴-𝐵 + 𝐻2𝑂 → 𝐴𝐻 + 𝐵𝑂𝐻 Examples: Lipase, choline esterase, acid and alkaline phosphates, pepsin, urease, rennin,

chymotrypsin, acyl choline acylhydrolase etc. Specific reactions:

i. Urease acts on urea.

𝐻2𝑁 − 𝐶𝑂 − 𝑁𝐻2 + 𝐻2𝑂𝒖𝒓𝒆𝒂𝒔𝒆→ 𝐶𝑂2 + 2𝑁𝐻3

ii. β-galactosidase acts on glycosyl compound.

𝛽-𝐷-galactoside +𝐻2𝑂𝜷−𝒈𝒂𝒍𝒂𝒄𝒕𝒐𝒔𝒊𝒅𝒂𝒔𝒆→ an alcohol + 𝐷-galactose

iii. Acyl choline acylhydrolase acts on exter bonds.

an acylcholine +𝐻2𝑂𝒂𝒄𝒚𝒍 𝒄𝒉𝒐𝒍𝒊𝒏𝒆 𝒂𝒄𝒚𝒍𝒉𝒚𝒅𝒓𝒐𝒍𝒂𝒔𝒆→ choline + an acid

D. Lyases: Reaction catalyzed: Catalyze the cleavage of 𝐶 − 𝐶, 𝐶 − 𝑆 and certain 𝐶 − 𝑁 bonds i.e.,

removal of groups (𝐻2𝑂,𝐶𝑂2,𝑁𝐻3) from substrates by mechanisms other than hydrolysis (leaving double bond).

𝐴-𝐵 + 𝑋-𝑌 → 𝐴𝑋-𝐵𝑌 Examples: Aldolase, fumarase, histidase, carbonic anhydrase, pyruvate decarboxylase etc. Specific reactions:

i. Pyruvate decarboxylase acts on pyruvic acid and splits it into acetaldehyde and 𝐶𝑂2. 𝐻3𝐶 − 𝐶 − 𝐶𝑂𝑂

−

|| 𝑂

𝒑𝒚𝒓𝒖𝒗𝒂𝒕𝒆 𝒅𝒆𝒄𝒂𝒓𝒃𝒐𝒙𝒚𝒍𝒂𝒔𝒆→

𝐻3𝐶 − 𝐶𝐻 || 𝑂

+ 𝐶𝑂2

ii. Carbonic anhydrase acts on carbonic acid and splits it into 𝐻2𝑂 and 𝐶𝑂2.

𝐻2𝐶𝑂3𝒄𝒂𝒓𝒃𝒐𝒏𝒊𝒄 𝒂𝒏𝒉𝒚𝒅𝒓𝒂𝒔𝒆→ 𝐻2𝑂+ 𝐶𝑂2

iii. Fumarase catalyze the malate to form fumarate.

𝐿-malate𝒇𝒖𝒎𝒂𝒓𝒂𝒔𝒆→ fumerate +𝐻2𝑂

E. Isomerases: Reaction catalyzed: Catalyze interconversion of optical, geometrical or positional isomers

i.e., isomerization reaction. 𝐴 → 𝐴′

Examples: Triose phosphate isomerase, ketoisomerase, retinol isomerase, phosphohexose isomerase, phosphoglucose isomerase etc.

Specific reactions: i. Phosphoglucose isomerase converts glucose to fructose.

glucose-6-phosphate𝒑𝒉𝒐𝒔𝒑𝒉𝒐𝒈𝒍𝒖𝒄𝒐𝒔𝒆 𝒊𝒔𝒐𝒎𝒆𝒓𝒂𝒔𝒆→ fructose-6-phosphate

ii. Methylmaloxy CoA mulase converts methylmaloxy CoA to succinyl CoA. 𝐶𝐻3 |

𝑂𝑂𝐶 − 𝐶𝐻 − 𝐶 − 𝐶𝑜𝐴 −

|| 𝑂

𝒎𝒆𝒕𝒉𝒚𝒍𝒎𝒂𝒍𝒐𝒙𝒚 𝑪𝒐𝑨 𝒎𝒖𝒍𝒂𝒔𝒆⇔

𝑂𝑂𝐶 − 𝐶𝐻2 − 𝐶𝐻2 − 𝐶 − 𝐶𝑜𝐴 −

|| 𝑂

F. Ligases or synthetases: Reaction catalyzed: Catalyze formation of bond between 𝐶 − 𝐶, 𝐶 − 𝑆 and 𝐶 − 𝑁 by the

hydrolysis of ATP (coupled to the breaking of a pyrophosphate bond in ATP or a similar compound).

𝐴 + 𝐵 − − − −⟶ 𝐴-𝐵

ATP ADP + Pi



Examples: Glutamine synthetase, acetyl CoA carboxylase, succinate thiokinase, pyruvate carboxylase etc.

Specific reactions: i. Pyruvate carboxylase catalyzes formation of 𝐶 − 𝐶 bond.

𝐻3𝐶 − 𝐶 − 𝐶𝑂𝑂−

|| 𝑂

+ 𝐶𝑂2𝒑𝒚𝒓𝒖𝒗𝒂𝒕𝒆 𝒄𝒂𝒓𝒃𝒐𝒙𝒚𝒍𝒂𝒔𝒆

→ 𝐻𝑂𝑂𝐶 − 𝐶𝐻2 − 𝐶 − 𝐶𝑂𝑂

−

|| 𝑂

Pyruvate ATP ADP Oxaloacetate

ii. Succinate thiokinase catalyzes formation of 𝐶 − 𝑆 bonds.

𝐺𝑇𝑃 + succinate + 𝐶𝑜𝐴𝒔𝒖𝒄𝒄𝒊𝒏𝒂𝒕𝒆 𝒕𝒉𝒊𝒐𝒌𝒊𝒏𝒂𝒔𝒆→ 𝐺𝐷𝑃 + 𝑃𝑖 + succinyl-𝐶𝑜𝐴

iii. Glutamine synthetase catalyzes formation of 𝐶 − 𝑁 bonds.

𝐴𝑇𝑃 + 𝐿-glutamate + 𝑁𝐻4+ 𝐠𝐥𝐮𝐭𝐚𝐦𝐢𝐧𝐞 𝐬𝐲𝐧𝐭𝐡𝐞𝐭𝐚𝐬𝐞→ 𝐴𝐷𝑃 + orthophosphate + 𝐿-glutamine

6. Write down the general properties of enzymes. Following are the properties of enzymes: a) Active site: Enzyme possesses an active site containing amino acid side chains – interaction

with substrate. b) Catalytic efficiency: Highly efficient, can increase the rate of reaction up to 103 − 108 times.

One enzyme molecule can react with 100 to 1000 molecules of substrate. c) Specificity: Highly reaction specific. d) Co-factors: Some enzymes associate with a non-protein co-factor (known as coenzyme) that is

needed for enzyme activity. e) Regulation: Enzymes can be inhibited or activated in their function.

When the product amount is low ⟶ enzymes work more

When the product amount is high ⟶ enzymes work less f) Location within the cell: Some are in the cytosol so they can only perform reaction in cytosol,

not in mitochondria or anywhere else. g) Other properties:

i. Catalyst of living world. ii. Protein in nature.

iii. Usually globular protein but some are crystalline. iv. Action is rapid and accurate. v. Heat labile.

vi. Non-dialyzable. vii. Colorless (some are yellow, blue, green) and soluble in water, acid solution, salt solution etc.

viii. Precipitates by protein precipitating agents like concentrated alcohol, ammonium sulphate, trichloroacetic acid etc.

ix. Lowers the activation energy. x. Don't initiate the reaction.

xi. Huge in size. xii. Small active complex.

xiii. Effective in very small amount. xiv. Neither altered / destroyed in the reaction. xv. Can be activated or inhibited by high temperature, 𝑝𝐻, UV rays, heavy metals etc.



7. What is the active site of the enzymes? Active site: The active site (or active center) of an enzyme represents as the small region at which

the substrates bind and participate in the catalysis. So, the portion of the enzyme protein molecule which actually takes part in catalysis is called the active site or catalytic site of the enzyme. If protein has active site it is enzyme otherwise protein. So, every enzyme is protein but every protein is not enzyme. Certain common features about the active sites are:

The active sites are regarded as clefts or crevices or pockets occupying a small volume of the total protein of an enzyme.

The active site is a three dimensional activity.

The specificity of the substrate binding depends upon the arrangement of the atoms or groups at the active site.

It is made up of groups that come from the different parts of the linear amino acids chain. Indeed the residues are far apart in the linear sequence but may come together to bring about catalysis.

The active site is not rigid in structure and shape. It is rather flexible to promote the specific substrate binding.

Generally, the active site possesses a substrate binding site and a catalytic site. The latter is for the catalysis of the specific reaction.

The coenzymes or cofactors on which some enzymes depend are present as a part of the catalytic site.

The substrates bind at the active site by weak non-covalent bonds.

Enzymes are specific in their action due to the existence of active sites.

The commonly found amino acids at the active sides are serine, aspartate, histidine, cysteine, lysine, arginine, glutamate, tyrosine etc. Among them serine is most frequently found.

The substrate [𝑆] binds the enzyme (𝐸) at the active site to form enzyme-substrate complex (𝐸𝑆). The product (𝑃) is released after the catalysis and the enzyme is available for reuse.

𝐸 + 𝑆 ⇔ 𝐸𝑆 → 𝐸 + 𝑃

8. Describe the mechanism of enzyme action. The enzyme catalyzed reaction obeys the general laws of chemistry as (i) the reactant (or

substrate) must be colloidal (ii) molecular collision must occur with the correct orientation (iii) reactant or substrate must have sufficient energy called activation energy. If two reactants colloid with each other having activation energy, then the reaction takes place.

Figure 1: Energy of activation diagram without and with enzyme



Enzyme reduces the activation energy of substrates. Enzymes can act in several ways to lower 𝛥𝐺. Such as

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.

There are many hypotheses to explain the mechanism of enzyme action. They are

a. Fisher theory: Emil Fisher in 1894 proposed that the union between the substrate and the enzyme takes place at the active state more or less in a manner in which a key fits a lock and results in the formation of an enzyme-substrate complex.

Figure 2: Fisher's lock-key model

b. Michaelis-Menten theory: The enzyme (𝐸) combines with its substrate (𝑆) to form enzyme-substrate complex (𝐸𝑆), within the complex the substrate breaks down into the product (𝑃) and then the enzyme dissociates from the substance.

𝐸 + 𝑆 ⇔ 𝐸𝑆 → 𝐸 + 𝑃 c. Koshland’s theory: In 1958 Daniel Koshland suggested a modification to the lock and key

model. An enzyme when occupied by the substrate has a particular geometrical shape. When enzyme is combined with a substrate the shape of the enzyme molecules altars slightly and because of these alterations the substrate can become fit properly into the enzyme molecules. In this alter configuration the enzyme is unstable.

Figure 3: Koshland's induced fit model

9. Write down the characteristics of enzyme actions. Enzyme action shows the following characteristics:

Specificity: An enzyme can catalyze one specific reaction by acting only on a particular type of substrate. For example, amylase acts on starch, lipase acts on lipid etc.



Enzymes cannot initiate a chemical reaction; they only stimulate or retard the process.

Enzymes only stimulate or retard the intermediate steps in the chemical process.

Enzymes are not destroyed in the process, as they are catalytic agent.

Every enzyme acts best at a limited range of 𝑝𝐻 and shows maximum activity at optimum 𝑝𝐻.

Every enzyme shows its maximum activity at a particular temperature.

Enzyme action is generally reversible.

A small amount of enzyme may act upon unlimited quantity of substrate.

10. Describe the factors that affect the enzymic activity. The following factors, which influence the rate of enzyme activity, have been summarized below: A. Substrate Concentration: It has been shown experimentally that if the amount of the enzyme

is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum. After this point, increases in substrate concentration will not

increase the velocity (Δ𝐴 Δ𝑇⁄ ). This is represented graphically in Figure.

Figure 4: Effect of substrate concentration

It is theorized that when this maximum velocity had been reached, the entire available enzyme has been converted to 𝐸𝑆, the enzyme substrate complex. This point on the graph is designated 𝑉𝑚𝑎𝑥. Using this maximum velocity

𝐸 + 𝑆𝐾+1⟸⟹𝐾−1

𝐸𝑆𝐾+2⟸⟹𝐾−2

𝑃 + 𝐸

Michaelis developed a set of mathematical expressions to calculate enzyme activity in terms of reaction speed from measurable laboratory data. The Michaelis constant Km is defined as the substrate concentration at 1/2 the maximum velocity. This is shown in Figure 8. Using this constant and the fact that Km can also be defined as:

𝐾𝑚 =𝐾+1 + 𝐾+2𝐾−1

= [𝑆]12𝑉𝑚𝑎𝑥

𝐾+1 , 𝐾−1 and 𝐾+2 being the rate constants from equation (7). Michaelis developed the following expression for the reaction velocity in terms of this constant and the substrate concentration.

𝑉 =𝑉𝑚𝑎𝑥[𝑆]

𝐾𝑚 + [𝑆]

Where,



𝑉 = The velocity at any time [𝑆] = The substrate concentration at this time 𝑉𝑚𝑎𝑥 = The highest under this set of experimental conditions (𝑝𝐻, temperature etc.) 𝐾𝑚 = The Michaelis constant for the particular enzyme being investigated Michaelis constants have been determined for many of the commonly used enzymes. The size

of Km tells us several things about a particular enzyme.

A small Km indicates that the enzyme requires only a small amount of substrate to become saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations.

A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity.

The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently assumed to be enzyme's natural substrate, though this is not true for all enzymes.

B. Effect of enzyme concentration: In order to study the effect of increasing the enzyme

concentration upon the reaction rate, the substrate must be present in an excess amount; i.e., the reaction must be independent of the substrate concentration. Any change in the amount of product formed over a specified period of time will be dependent upon the level of enzyme present. Graphically this can be represented as:

Figure 5: Zero order reaction rate is independent of substrate concentration

These reactions are said to be "zero order" because the rates are independent of substrate concentration and are equal to some constant 𝑘. The formation of product proceeds at a rate which is linear with time. The addition of more substrate does not serve to increase the rate. In zero order kinetics, allowing the assay to run for double time results in double the amount of product.

Reaction Orders with Respect to substrate Concentration:

Order Rate equation Comments

Zero Rate = 𝑘 Rate is independent of substrate concentration

First Rate = 𝑘[𝑆] Rate is proportional to the first power of substrate concentration

Second Rate = 𝑘[𝑆][𝑆] = 𝑘[𝑆]2 Rate is proportional to the square of the substrate concentration

Second Rate = 𝑘[𝑆1][𝑆2] Rate is proportional to the first power of each of two reactants

The amount of enzyme present in a reaction is measured by the activity it catalyzes. The relationship between activity and concentration is affected by many factors such as temperature, pH, etc. An enzyme assay must be designed so that the observed activity is proportional to the amount of enzyme present in order that the enzyme concentration is the only limiting factor. It is satisfied only when the reaction is zero order.

In Figure 6, activity is directly proportional to concentration in the area AB, but not in BC. Enzyme activity is generally greatest when substrate concentration is unlimiting.



Figure 6: Activity vs concentration

When the concentration of the product of an enzymatic reaction is plotted against time, a similar curve results, Figure 7.

Figure 7: Reaction rate limited by substrate concentration

Between A and B, the curve represents a zero order reaction; that is, one in which the rate is constant with time. As substrate is used up, the enzyme's active sites are no longer saturated, substrate concentration becomes rate limiting, and the reaction becomes first order between Band C. To measure enzyme activity ideally, the measurements must be made in that portion of the curve where the reaction is zero order. A reaction is most likely to be zero order initially since substrate concentration is then highest. To be certain that a reaction is zero order; multiple measurements of product (or substrate) concentration must be made.

C. Effect on the 𝒑𝑯 of the solution: Enzymes are affected by changes in 𝑝𝐻. The most favorable 𝑝𝐻 value - the point where the enzyme is most active - is known as the optimum 𝑝𝐻 (4 − 7). This is graphically illustrated in Figure 8.

Figure 8: Effect of pH on reaction rate



Extremely high or low 𝑝𝐻 values generally result in complete loss of activity for most enzymes. 𝑝𝐻 is also a factor in the stability of enzymes. The variation in 𝑝𝐻 may influence the ionization of different groups in the binding and catalytic sites of the enzyme and also in the substrate. As with activity, for each enzyme there is also a region of 𝑝𝐻 optimal stability. The optimum 𝑝𝐻 value will vary greatly from one enzyme to another. 𝑝𝐻 for Optimum Activity:

Enzyme 𝑝𝐻 optimum

Lipase (pancreas) 8.0

Lipase (stomach) 4.0 − 5.0

Lipase (castor oil) 4.7

Pepsin 1.5 − 1.6

Trypsin 7.8 − 8.7

Urease 7.0

Invertase 4.5

Maltase 6.1 − 6.8

Amylase (pancreas) 6.7 − 7.0

Amylase (malt) 4.6 − 5.2

Catalase 7.0

D. Effect of the temperature of the solution: Like most chemical reactions, the rate of an enzyme-

catalyzed reaction increases as the temperature is raised. For each enzyme there is a particular temperature which is called optimum temperature at which the enzyme activity is maximum and the activity progressively declined both above and below this temperature. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results. In the case of enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by high temperatures. As shown in Figure 13, the reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. Because most animal enzymes rapidly become denatured at temperatures above 40℃, most enzyme determinations are carried out somewhat below that temperature.

Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5℃ or below is generally the most suitable. Some enzymes lose their activity when frozen.

Figure 9: Effect of temperature on reaction rate

E. Effect of product concentration: The concentration of the end products of an enzymic action has a retarding effect upon the rate of enzyme action. The explanation of retarding the reaction rate lays on that a more stable complex of enzyme and reaction product forms than that of enzyme and substrate.



F. Effect of modifiers: Some of the enzymes require certain activators. Activators increase enzyme velocity through various mechanisms – combining with the substrate, formation of 𝐸𝑆-metal complex, direct participation in the reaction and bringing a conformational change in the enzyme.

The facts are (i) presence of inorganic metals e.g. 𝑀𝑔++, 𝐹𝑒++ , 𝑍𝑛++ , 𝐶𝑢++ , 𝑀𝑛++ etc. increases the catalytic activity of enzyme, (ii) presence of mercury salt, gold, silver etc. decrease the catalytic activity of enzyme, (iii) rarely, anions are also needed for enzyme activity e.g. chloride ion (𝐶𝑙−) for amylase, (iv) formaldehyde destroys enzymes.

G. Effect of coenzyme or cofactor: Enzymes (excepting the enzymes of GIT), work efficiently in

presence of coenzymes and cofactors e.g. 𝑀𝑔++, 𝐶𝑎++ etc. H. Effect of hormones: Many hormones can influence the enzyme activity. For example, glucagon

and epinephrine enhance the production of cyclic 𝐴𝑀𝑃 in the liver cells. The cyclic 𝐴𝑀𝑃 in turn promotes the conversion of enzyme phosphorylase into its active form.

I. Effect of light and radiation: Enzymes are highly sensitive to short wavelength (high energy)

radiation such as x-ray, UV, beta, gamma rays. They form peroxides which cause oxidation of the enzyme resulting loss in enzyme activity e.g. UV rays inhibit salivary amylase activity.

J. Effect of inhibitor: Enzyme inhibitors are substances which alter the catalytic action of the

enzyme and consequently slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition - competitive, non-competitive and substrate inhibition.

Competitive inhibition : Competitive inhibition occurs when the substrate and a substance resembling the substrate are both added to the enzyme. A theory called the "lock-key theory" of enzyme catalysts can beused to explain why inhibition occurs.

Figure 10: Lock-key theory - competitive inhibitor

The lock and key theory utilizes the concept of an "active site." The concept holds that one particular portion of the enzyme surface has a strong affinity for the substrate. The substrate is held in



such a way that its conversion to the reaction products is more favorable. If we consider the enzyme as the lock and the substrate the key (Figure 10) - the key is inserted in the lock, is turned, and the door is opened and the reaction proceeds. However, when an inhibitor which resembles the substrate is present, it will compete with the substrate for the position in the enzyme lock. When the inhibitor wins, it gains the lock position but is unable to open the lock. Hence, the observed reaction is slowed down because some of the available enzyme sites are occupied by the inhibitor. If a dissimilar substance which does not fit the site is present, the enzyme rejects it, accepts the substrate, and the reaction proceeds normally.

Non-competitive inhibitor: Non-competitive inhibitors are considered to be substances which when added to the enzyme alter the enzyme in a way that it cannot accept the substrate.

Figure 11: Non-competitive inhibitor

Substrate inhibition: Substrate inhibition will sometimes occur when excessive amounts of substrate are present. Figure 12 shows the reaction velocity decreasing after the maximum velocity has been reached.

Figure 12: Substrate become rate inhibiting

Additional amounts of substrate added to the reaction mixture after this point actually decreases the reaction rate. This is thought to be due to the fact that there are so many substrate



molecules competing for the active sites on the enzyme surfaces that they block the sites (Figure 13) and prevent any other substrate molecules from occupying them.

Figure 13: Substrate inhibition

This causes the reaction rate to drop since the entire enzyme present is not being used. In addition to temperature and pH there are other factors, such as ionic strength, which can affect

the enzymatic reaction. Each of these physical and chemical parameters must be considered and optimized in order for an enzymatic reaction to be accurate and reproducible.

11. What do you know about specificity of enzyme action? What do you mean by enzyme specificity? Except few enzymes they are specific in their action. A specific enzyme can act upon a particular

substrate. E.g. zymase acts upon only glucose to give alcohol and 𝐶𝑂2, lactase acts on milk etc. Thus the enzymes may exhibit in different types of specificities, which are given below:

a) Reaction specificity, b) Linkage specificity, c) Stereospecificity or optical specificity, d) Substrate specificity and e) Kinetic specificity.

a. Reaction specificity: The same substrate can undergo different types of reactions, each

catalyzed by a separate enzyme and this is referred to as reaction specificity. An amino acid can undergo transamination, oxidative deamination, decarboxylation, racemization etc. The enzymes however, are different for each of these reactions.

𝐻 |

𝑅 − 𝐶 − 𝐶𝑂𝑂𝐻| 𝑁𝐻2

𝒐𝒙𝒊𝒅𝒂𝒔𝒆→

𝑂 ||

𝑅 − 𝐶 − 𝐶𝑂𝑂𝐻

𝐻 |

𝑅 − 𝐶 − 𝐶𝑂𝑂𝐻| 𝑁𝐻2

𝒅𝒆𝒄𝒂𝒓𝒃𝒐𝒙𝒚𝒍𝒂𝒔𝒆𝒆→ 𝑅 − 𝐶𝐻2 − 𝑁𝐻2 + 𝐶𝑂2



b. Linkage specificity: Some enzymes split particular bonds between amino acids in peptide fragments which are used to determine amino acid sequence in protein. Thus enzymes are linkage specific.

c. Stereospecificity or optical specificity: Stereoisomers are the compounds which have the same molecular formula, but differ in their structural configuration. Some enzymes act only on one isomer and, therefore, exhibit stereospecificity. The class of enzymes belonging to isomerases does not exhibit stereospecificity, since they are specialized in the interconversion of isomers.

For example, L-amino acid oxidase, a flavin enzyme will act on a L-isomer only but not on a D-isomer.

𝐻 |

𝑅 − 𝐶 − 𝐶𝑂𝑂−

|

𝑁𝐻3+

+ 𝑂2 +𝐻2𝑂𝑳−𝒂𝒎𝒊𝒏𝒐 𝒂𝒄𝒊𝒅 𝒐𝒙𝒊𝒅𝒂𝒔𝒆→

𝑂 ||

𝑅 − 𝐶 − 𝐶𝑂𝑂− +𝑁𝐻4+ + 𝐻2𝑂2

D-amino acid oxidase acts on D-amino acids. Hexokinase acts on D-hexoses. Glucokinase acts on D-glucose. Amylase acts on α-glycosidic linkages. Cellulase cleaves β-glycosidic bonds. Maltase hydrolyses α-glucosides. Stereospecificity is explained by considering three distinct regions of substrate molecule

specifically binding with three complementary regions on the surface of the enzyme.

Figure 14: Diagrammatic representation of stereospecificity (a', b', c') – three-point attachment of substrate to the enzyme (a, b, c).

d. Substrate specificity: The substrate specificity varies from enzyme to enzyme. It may be absolute, relative or broad.

i. Absolute substrate specificity: Certain enzymes act only on one particular substrate. For example, urease acts only on urea to produce ammonia and 𝐶𝑂2.

𝐻2𝑁 − 𝐶𝑂 − 𝑁𝐻2 + 𝐻2𝑂𝒖𝒓𝒆𝒂𝒔𝒆→ 𝐶𝑂2 + 2𝑁𝐻3

ii. Relative substrate specificity: Some enzymes act on structurally related substances. This, in turn, may be dependent on the specific group or a bond present. The action of trypsin is a good example for group specificity. Trypsin hydrolyses peptide linkage involving arginine or lysine. Chymotrypsin cleaves peptide bonds attached to aromatic amino acids (phenylalanine, tyrosine and tryptophan). Examples of bond specificity – glycosidases acting on glycosidic bonds of carbohydrates, lipases cleaving ester bonds of lipids etc.

iii. Broad substrate specificity: Some enzymes act on closely related substrates which is commonly known as broad substrate specificity. E.g. hexokinase acts on hexoses (glucose, fructose,

mannose, glucosamine but not on galactose). It is possible that some structural similarity among the first four compounds makes them a common substrate for the enzyme hexokinase.



glucose

fructose} + 𝐴𝑇𝑃

𝒉𝒆𝒙𝒐𝒌𝒊𝒏𝒂𝒔𝒆→

glucose-6-phosphate

fructose-6-phosphate} + 𝐴𝐷𝑃

e. Kinetic specificity: Many enzymes exhibit a kinetic specificity. E.g. esterase, although hydrolyzing all esters but the rate of hydrolysis is different for different esters. The amino group belongs to an aromatic amino group and for the carboxylic group in one of the dicarboxylic amino acid.

12. Give your understanding about folding / active site formation of enzyme. When enzyme is dissolved in water, then enzyme is folded. This phenomenon is called folding of

enzyme. Folding of enzyme is caused by three kinds of factor:

Hydrogen bonds,

Electrostatic bonds (hydrophilic and hydrophobic) and

Van der Waals bonds. Hydrogen bonds: Enzymes are the polymer of amino acids. In an enzyme chain >𝑁𝐻 and >𝐶𝑂

groups work in 𝛼-helix and 𝛽-sheets. In fact side chains of 11 to 20 fundamental amino acids can also

participate in hydrogen bonding. 𝛼-helix and 𝛽-sheets are stabilized by the interaction of hydrogen bond between >𝑁𝐻 and >𝐶𝑂 groups on adjacent chain. Folding in enzyme can be broken by adding urea and then enzyme is formed in general sequence.

𝛼-helix 𝛽-sheets Electrostatic bonds (hydrophilic and hydrophobic): Enzyme contains two parts in its molecule

namely hydrophilic and hydrophobic parts. When an enzyme is taken in water, at this every moment hydrophilic group is attacked by water molecules and on the other hand the hydrophobic group is repulsed by water molecules occurring an interaction among them. Just at the very moment the enzyme molecules are folded.

Van der Waals forces: When molecules are taken into water then these are divided into two parts

namely polar and non-polar parts. Almost all the polar parts remain on the surface except very few polar parts. Non-polar parts are precipitated into water. Not all the non-polar residues remain inside,



some especially shorter ones (glycine, alanine) are on the surface. The non-polar residues almost everywhere close-packed in Van der Waals contact with their neighbor bond. Water molecules are attracted to all polar groups on the surface including main chain and >𝑁𝐻 groups. About 75% of the

amino acid residues are arranged in the form of right handed 𝛼-helix both in crystal and solution. Mechanism: Resists the approach to each of all the atom not actually bonded when these

approach less than a minimum distance (5.4 × 10−8 𝑐𝑚).

13. What do you mean by enzyme modifiers? Enzyme modifier: The agent that modifies the catalytic activity of the enzyme are known as

enzyme modifiers. Enzyme modifiers are two types:

Positive modifiers: The agents which increase the catalytic activity of enzyme are known as positive modifiers. Examples: inorganic metal ions e.g. 𝐹𝑒++,𝑀𝑔++, 𝑍𝑛++, 𝐶𝑢++, 𝑀𝑛++ etc.

Negative modifiers: The agents which decrease the catalytic activity of enzyme are known as negative modifiers. Examples: mercury, silver, gold, chloroform etc.

14. What is inhibitor? Enumerate the name of common inhibitor available in the market. Inhibitor: Inhibitor is defined as any substance that can diminish the velocity of an enzyme

catalyzed reaction. Inhibitor binds with enzyme and brings about a decrease in catalytic activity of that enzyme. The inhibitor may be organic or inorganic in nature. It blocks enzyme activity by two types namely 𝐶𝑎-channel blocker and 𝛽-blocker.

Inhibitors are of three types. They are:

Competitive: These resemble real substrate and compete with the substrate to combine with the catalytic sites of the enzymes.

𝐸+𝑆↔𝐸𝑆 ⟶ 𝐸 + 𝑃

↓ +𝐼 𝐸𝐼 ↛ 𝑃

Non-competitive: These do not compete for the active sites. They combine with either free enzyme or enzyme-substrate complexes.

𝐸 + 𝑆 ⟺ 𝐸𝑆 ⟶ 𝐸 + 𝑃+ + 𝐼 𝐼 ↕ ↕

𝐸𝐼 + 𝑆 𝐸𝐼𝑆

Uncompetitive: Not very common. Only combines with enzyme-substrate complex.



The name of common inhibitors available in the market and significance of these inhibitors are as follows:

Inhibitor Significance of inhibitor

Amlodipine (BEX) Camlodine Sq)

Used for controlling blood pressure.

Ramipril (Sq) Used for controlling blood pressure.

Tenorin ACI) Used as 𝛽-blocker.

Aminopterin Amethoptein Methotrexate

Used in the treatment for leukemia and other cancers.

Succinyl chloride Used in surgery for muscle relaxation in anaesthetized patients.

Ephidrena Amphetamine

Used for elevating catcholamine levels.

Dicumarol Hydrazide (INH)

Acts as an anticoagulant. Used leads to 𝐵6 defficiency.

Allopurinol Used in the treatment of gout.

15. What do you mean by substrate? Substrate: Substrate means the main chemical compound which undergoes alteration in a

chemical reaction upon which the enzyme exerts its influence.

16. Define apoenzyme, coenzyme, cofactor and holoenzyme. Apoenzyme: The protein part of a holoenzyme is called apoenzyme. So the enzyme without the co-

factor is called apoenzyme or when enzyme does not process the co-factor, the remaining conjugated proteins are called the apoenzyme. These types of enzymes are inactive.

Coenzyme: Many enzymes catalyze reactions of their substrates only in the presence of specific

heat-stable, low molecular weight organic molecules called coenzyme. We get a factor or group without amino acid when we hydrolyze protein. This factor is called coenzyme. The coenzyme e.g. 𝐾+, 𝐹𝑒3+ etc. activates apoenzyme.

Coenzymes are heat-stable, dialyzable, non-protein organic molecules and the prosthetic groups of enzymes.

Coenzymes are small organic molecules that transport chemical groups from one enzyme to another. Some of these chemicals such as riboflavin, thiamine and folic acid are vitamins, this is when these compounds cannot be made in the body and must be acquired from the diet. The chemical groups carried include the hydride ion (𝐻−) carried by 𝑁𝐴𝐷 or 𝑁𝐴𝐷𝑃+, the acetyl group carried by coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by S-adenosylmethionine.

Figure 15: Space-filling model of the coenzyme NADH



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.

Coenzymes are usually 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.

Cofactor: 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. 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 𝐴𝑇𝑃. These molecules act to transfer chemical groups between enzymes.

Figure 16: Ribbon-diagram showing carbonic anhydrase II. The grey sphere is the zinc cofactor in the active site

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. 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.

Holoenzyme: Inactive apoenzymes are activated by the cofactor; this activated enzyme is called

holoenzyme. In a word catalytically active enzyme with its cofactor is called holoenzyme. The term holoenzym 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.

Relations: 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., thiamine pyrophosphate in the enzyme pyruvate dehydrogenase). Therefore,

Apoenzyme + Coenzyme/cofactor → Holoenzyme Antienzyme: Antienzymes are the substances produced as a result of repeated injection of certain

enzymes in the serum, which can prevent the normal action of enzymes injected e.g. anti-pepsin, anti-tripsin, anti-renin etc.



17. Differentiate between enzyme and coenzyme. Followings are the differences between enzyme and coenzyme:

Enzyme Coenzyme

Enzyme is a organic catalyst. Coenzyme is accessory substances.

Enzyme is protein in nature. Coenzyme is non-protein in nature.

Enzyme is non-dialyzable. Coenzyme is dialyzable.

Enzyme is heat and 𝑝𝐻 liable. Coenzyme is heat and 𝑝𝐻 stable.

Enzyme can act itself. Coenzyme is required by an enzyme for its catalytic reaction.

18. What is the difference between prosthetic group and coenzyme? Prosthetic group is tightly bound to the enzyme, whereas coenzymes exist in the solution in free

state and contact the enzyme only at the time of reaction.

19. What do you mean by activators? What are metals that act as coenzyme? Activators: In some instances, the enzyme action is activated by some metallic ions, known as

activators. Metals that act as coenzymes are: i. 𝑍𝑛++ ion for carbonic anhydrase,

ii. 𝐶𝑢++ ion for tyrosine, iii. 𝐹𝑒++ ion for catalase peroxidase, iv. 𝑀𝑔++ ion for pyrophosphatase and v. 𝑀𝑛++ ion for aminopeptidase.

20. Distinguish between hormone & enzyme. Differences between hormone and enzyme are given below:

Hormone Enzyme

Hormone is a chemical substance that is secreted into the body fluid by one cell or a group of cells and that exert physiological control effect on other cells of the body.

It is an organic catalyst produces by the living cell.

Hormone is steroid, peptide, protein or amino acid in nature.

Enzyme is protein in nature.

Hormone secretion depends upon releasing or inhibitory hormone from the hypothalamus.

Enzyme activity depends upon substrate concentration, 𝑝𝐻, temperature.

Controlled by (secretion or production) feedback mechanism.

Controlled by hormone, physical or chemical effects.

It does not act as a catalyst. Enzyme acts as a catalyst.

Transported in the blood throughout the body and acts far from the site of origin.

Acts at the site where it is produced but some (e.g. digestive enzymes) function outside their cell of origin.

Hormone exhibits a high degree of specificity of action, only target cells will respond to their hormones.

Enzyme exhibits a relative or absolute specificity of action.



21. Discuss the uses of enzymes as therapeutic agents. Enzymes as therapeutic agents:

Streptokinase prepared from streptococcus is useful for clearing the blood clots. Streptokinase activates plasma plasminogen to plasmin which, in turn, attacks fibrin to convert into soluble products.

Plasminogen

↓ 𝑆𝑡𝑟𝑒𝑝𝑡𝑜𝑘𝑖𝑛𝑎𝑠𝑒 Plasmin ↓

Fibrin (clot) → Solubleproducts

The enzyme aparaginase is used in the treatment of leukemias. Tumor cells are dependent on asparagine of the host’s plasma for their multiplication. By administering asparaginase, the host’s plasma levels of asparagine are drastically reduced. This leads to depression in the viability of tumor cells.

Enzyme is used in heart stimulating injection such as inj. digitoxin, inj. digotoxic etc. It is made from cardiac glycoside which is extracted from plant digitalis.

Following a list of therapeutic application of enzymes is given:

Enzyme Therapeutic application

Streptokinase/urokinase To remove blood clot.

Asparaginase In cancer therapy.

Papain Anti-inflammatory.

𝛼1-antitrypsin To treat emphysema (breathing difficulty due to distension of lungs).

22. Discuss diagnostic importance of enzyme. What are the clinical diagnoses of enzymes? Estimation of enzyme activities in biological fluids (particularly plasma/serum) is of great clinical

importance. Enzymes in the circulation are divided into two groups – plasma functional and plasma non-functional.

Estimation of the activities of plasma non-functional enzymes is very important for the diagnosis and prognosis of several diseases.

The normal serum level of an enzyme indicates the balance between its synthesis and release in the routine cell turnover. The raised enzyme levels could be due to cellular damage, increased rate of cell turnover, proliferation of cells, increased synthesis of enzymes etc. Serum enzymes are conveniently used as markers to detect the cellular damage which ultimately helps in the diagnosis of diseases.

A summary of the important enzymes useful for the diagnosis of specific diseases is given in following table:

Serum enzyme (elevated) Disease (most important)

Amylase Acute pancreatitis

Serum glutamate pyruvate transaminase (SGPT) Liver diseases (hepatitis)

Serum glutamate oxaloacetate transaminase (SGOT) Heart attacks (myocardial infarction)

Alkaline phosphatase Rickets, obstructive jaundice

Lactate dehydrogenase (LDH) Heart attacks, liver diseases

Creatine phosphokinase (CPK) Myocardial infarction (early marker)

Alsolase Muscular dystrophy

5’-neucleotidase Hepatitis

𝛾-glutamyl transpeptidase (GGT) Alcoholism



Detailed information on the diagnostic enzymes including reference values is provided in following table:

Enzymes Reference value Disease(s) in which increased

Digestive enzymes: Amylase Lipase

2.5 − 5.5 𝜇𝐾𝑎𝑡

0.2 − 1.5 𝐼𝑈/𝐿

Acute pancreatitis, mumps (acute parotitis), obstruction in pancreatic duct, severe diabetic ketoacidosis. Acute pancreatitis, moderate elevation in carcinoma of pancreas.

Transaminases: Alanine transminase (ALT) or SGPT Asparatate transminase (AST) or SGOT

3 − 40 𝐼𝑈/𝐿

4 − 45 𝐼𝑈/𝐿

Acute hepatitis (viral or toxic), jaundice, cirrhosis of liver. Myocardial infarction, liver diseases, liver cancer, cirrhosis of liver.

Phosphatases: Alkaline phosphatase (ALP) Acid phosphatase (ACP)

In adults 25 − 90 𝐼𝑈/𝐿

2.5 − 12 𝐼𝑈/𝐿

Bone diseases (related to higher osteoblastic activity) – rickets, Pagets’ disease, hyperparathyroidism, carcinoma of bone. Prostatic carcinoma i.e. cancer of prostate gland, Pagets’ disease.

Enzymes of carbohydrate metabolism: Aldolase Isocitrate dehydrogenase (ICD) Lactate dehydrogenase (LDH)

2 − 6 𝐼𝑈/𝐿

1 − 4 𝐼𝑈/𝐿

50 − 200 𝐼𝑈/𝐿

Mascular dystrophy, liver diseases, myocardial infarction, myasthenia gravis, leukemias. Liver diseases (inflammatory toxic or malignant). Myocardial infarction, acute infective hepatitis, macular dystrophy, leukemia, pernicious anemia.

Miscellaneous enzymes: Creatine kinase (CK) or creatine phosphokinase (CPK) Choline esterase (ChE I) 5’-neucleotidase or nucleotide phosphatase (NTP) 𝛾-glutamyl transpeptidase (GGT) Ceruloplasmin (ferrooxidase)

10 − 50 𝐼𝑈/𝐿

2 − 10 𝐼𝑈/𝐿 2 − 15 𝐼𝑈/𝐿

5 − 40 𝐼𝑈/𝐿

20 − 50 𝑚𝑔/𝑑𝐿

Myocardial infarction, muscular dystrophy, hypothyroidism, alcoholism. Nephritic syndrome, myocardial infarction. Hepatitis, obstructive jaundice, tumors. Alcoholism, infective hepatitis, obstructive jaundice. Bacterial infections, collagen diseases, cirrhosis, pregnancy.

In following table decreased plasma enzymes in certain disorders are given:

Enzyme Reference values Disease(s) in which decreased

Amylase 80 − 180 𝑆𝑜𝑚𝑜𝑔𝑦𝑖 𝑢𝑛𝑖𝑡𝑠/𝑑𝐿

Liver diseases.

Pseudocholine esterase (ChE II)

10 − 20 𝐼𝑈/𝑑𝐿 Viral hepatitis, malnutrition, liver cancer, cirrhosis of liver.

Ceruloplasmin 20 − 50 𝑚𝑔/𝑑𝐿 Wilson’s disease.



Glucose 6-phosphate dehydrogenase (G6PD) in RBC

120 − 260 𝐼𝑈/1012 RBC Congenital deficiency with hemolytic anemia

23. What is pancreatin? Pancreatin: Pancreatin is a mixture of several digestive enzymes produced by exocrine cells of the

pancreas. It is composed of amylase, lipase and protease. This mixture is used to treat conditions in which pancreatic secretion are deficient. It is given in mouth in the treatment of pancreatic deficiency associated with condition such as cystic fibrosis and pancreatitis. It is denatured by acid and high doses, in entericoated dosage form, must be given to overcome the 𝑝𝐻 of the stomach.

Pancreatin hydrolyses fat, glycerol and fatty acids; break down proteins into peptides, proteases and derived substances and converts starch into dextrin and sugar. It is available in the market in the form of powder, capsules, containing powder on entericoated granules.

24. Describe your understanding about enzymatic hydrolysis of proteins. The dietary protein first reaches in the stomach where it is hydrolyzed by pepsin. Pepsin hydrolyses

the dietary protein into a mixture of polypeptide.

Dietary protein𝑝𝑒𝑝𝑠𝑖𝑛→ Polypeptide

The polypeptides formed thus in the stomach are digested in the intestine by tripsin, chymotrypsin and carboxy peptidases secreted in pancreatic juice and amino peptidases present in the intestinal mucosa. Tripsin hydrolyses peptide linkage containing arginine or lysine and chymotrypsin hydrolyses peptide linkages containing tyrosine or phenylalanine.

Polypeptides𝑡𝑟𝑦𝑝𝑠𝑖𝑛 𝑎𝑛𝑑 𝑐ℎ𝑦𝑚𝑜𝑡𝑟𝑦𝑝𝑠𝑖𝑛→ Peptides + Amino acids

Carboxy peptidase-𝐴 hydrolyses the end group of peptides containing aromatic or aliphatic amino acid and releases free amino acids. Carboxy peptidase-𝐵 hydrolyses peptides containing arginine and lysine residues. The intestinal mucosa also contains tripeptidase, dipeptidase etc. which hydrolyze tri-peptide and di-peptide respectively.

Peptides𝑐𝑎𝑟𝑏𝑜𝑥𝑦 𝑝𝑒𝑝𝑡𝑖𝑑𝑎𝑠𝑒→ Amino acids

Peptides𝑎𝑚𝑖𝑛𝑜𝑝𝑒𝑝𝑡𝑖𝑑𝑎𝑠𝑒→ Amino acids

Di-peptides𝑑𝑖𝑝𝑒𝑝𝑡𝑖𝑑𝑎𝑠𝑒→ Amino acids

Tri-peptides𝑡𝑟𝑖𝑝𝑒𝑝𝑡𝑖𝑑𝑎𝑠𝑒→ Amino acids

Thus the final products of digestion of protein are amino acids which are absorbed in the body.

25. Discuss the role of enzyme in the process of fermentation to produce alcohol. Enzymes play a great important role to produce alcohol in the process of fermentation. In this

process the enzyme diastase is used to hydrolyze starch to maltose. To the solution of maltose yeast is added. The enzymes maltase and zymase are present in the yeast. The enzyme maltase converts maltese into glucose and zymase converts glucose into alcohol.

2(𝐶6𝐻10𝑂5)𝑛 + 𝑛𝐻2𝑂𝑑𝑖𝑎𝑠𝑡𝑎𝑔𝑒→ 𝑛(𝐶12𝐻22𝑂11)

Maltose𝑚𝑎𝑙𝑡𝑎𝑠𝑒→ Glucose + Fructose

Glucose𝑧𝑦𝑚𝑎𝑠𝑒→ Alcohol + 𝐶𝑂2



26. Mention some industrial applications of enzymes. Enzymes are used in the chemical industry and other industrial applications when extremely

specific catalysts are required. Followings are the industrial applications of enzymes:

Application Enzymes used Uses

Food processing

Amylases from fungi and plants

Production of sugars from starch, such as in making high-fructose corn syrup. In baking, catalyze breakdown of starch in the flour to sugar. Yeast fermentation of sugar produces the carbon dioxide that raises the dough.

Proteases Biscuit manufacturers use them to lower the protein level of flour.

Baby foods Trypsin To predigest baby foods.

Brewing industry

Enzymes from barley are released during the mashing stage of beer production

They degrade starch and proteins to produce simple sugar, amino acids and peptides that are used by yeast for fermentation.

Industrially produced barley enzymes

Widely used in the brewing process to substitute for the natural enzymes found in barley.

Amylase, glucanases, proteases

Split polysaccharides and proteins in the malt.

Betaglucanases and arabinoxylanases

Improve the wort and beer filtration characteristics.

Amyloglucosidase and pullulanases

Low-calorie beer and adjustment of fermentability.

Proteases Remove cloudiness produced during storage of beers.

Acetolactatedecarboxylase (ALDC)

Increases fermentation efficiency by reducing diacetyl formation.

Fruit juices Cellulases, pectinases Clarify fruit juices.

Jeans fading Per oxidase In pumic wash.

Dairy industry

Rennin, derived from the stomachs of young ruminant animals (like calves and lambs)

Manufacture of cheese, used to hydrolyze protein.

Microbially produced enzyme

Now finding increasing use in the dairy industry.

Lipases Is implemented during the production of Roquefort cheese to enhance the ripening of the blue-mould cheese.

Lactases Break down lactose to glucose and galactose.

Meat tenderizers Papain To soften meat for cooking.

Starch industry

Amylases, amyloglucosideases and glucoamylases

Converts starch into glucose and various syrups.

Glucose isomerase

Converts glucose into fructose in production of high fructose syrups from starchy materials. These syrups have enhanced sweetening properties and lower calorific values than sucrose for the same



level of sweetness.

Paper industry Amylases, Xylanases, Cellulases and ligninases

Degrade starch to lower viscosity, aiding sizing and coating paper. Xylanases reduce bleach required for decolorising; cellulases smooth fibers, enhance water drainage, and promote ink removal; lipases reduce pitch and lignin-degrading enzymes remove lignin to soften paper.

Biofuel industry Cellulases

Used to break down cellulose into sugars that can be fermented (see cellulosic ethanol).

Ligninases Use of lignin waste.

Biological detergent

Primarily proteases, produced in an extracellular form from bacteria

Used for presoak conditions and direct liquid applications helping with removal of protein stains from clothes.

Amylases Detergents for machine dish washing to remove resistant starch residues.

Lipases Used to assist in the removal of fatty and oily stains.

Cellulases Used in biological fabric conditioners.

Contact lens cleaners

Proteases To remove proteins on contact lens to prevent infections.

Rubber industry Catalase To generate oxygen from peroxide to convert latex into foam rubber.

Photographic industry

Protease (ficin) Dissolve gelatin off scrap film, allowing recovery of its silver content.

Molecular biology Restriction enzymes, DNA ligase and polymerases

Used to manipulate DNA in genetic engineering, important in pharmacology, agriculture and medicine. Essential for restriction digestion and the polymerase chain reaction. Molecular biology is also important in forensic science.

Soap and detergent industry

Lipase Used to assist in the removal of fatty and oily stains.

Amylase Detergent for machine dish washing to remove resistance starch residues.

Cellulase Used in biological fabric conditions.

27. What are the biological roles of enzymes? The enzymes have many applications in our daily life. The manifold applications are described

below: i. Enzymes serve a wide variety of functions inside living organisms. They are indispensable for

signal transduction and cell regulation, often via kinases and phosphatases. They also generate movement, with myosin hydrolysing ATP to generate muscle contraction and also moving cargo around the cell as part of the cytoskeleton. Other ATPases in the cell membrane are ion pumps involved in active transport.

ii. Enzymes are also involved in more exotic functions, such as luciferase generating light in fireflies. Viruses can also contain enzymes for infecting cells, such as the HIV integrase and reverse transcriptase or for viral release from cells, like the influenza virus neuraminidase.

iii. An important function of enzymes is in the digestive systems of animals. Enzymes such as amylases and proteases break 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 hydrolyse 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 a herbivorous diets, microorganisms in the gut produce another enzyme, cellulase to break down the cellulose cell walls of plant fiber.

iv. 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 being provided by one enzyme but an inducible high activity from a second enzyme.

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

vi. Enzymes are used in the treatment of blood clot. Example: urokinase is used in the treatment of blood clot in brain.

vii. Enzymes help to make building tissues. viii. Enzymes take part in energy producing reactions.

ix. Cheese making: Enzymes play an important role in cheese making. They help in coagulating the casein in milk.

x. The enzymes are being used for processing fruits juice such as apple juice, grape juice etc. xi. Enzymes help in leather processing by de-haring hide. xii. Certain enzymes are used in chemical analysis. Example: urease is used for determination of

urea in blood. xiii. Enzymes play an important role in fermentation process to produce alcohol.

28. What are the physiological roles of enzyme? Followings are the physiological roles of enzymes:

i. Enzymes play an important in role in protein hydrolysis. The enzymes involved in protein hydrolysis are trypsin, peptidese, amino peptidese etc.

ii. Oxidoreductase enzymes are involved biological oxidation and reduction. For example dehydrogenase enzyme catalyses the removal of hydrogen and vice versa.

𝐶𝐻3 − 𝐶𝐻 − 𝐶𝑂𝑂−

| 𝑂𝐻

+ 𝑁𝐴𝐷+𝒍𝒂𝒄𝒕𝒂𝒕𝒆 𝒅𝒆𝒉𝒚𝒅𝒓𝒐𝒈𝒆𝒏𝒂𝒔𝒆↔

𝐶𝐻3 − 𝐶 − 𝐶𝑂𝑂−

|| 𝑂

+ 𝑁𝐴𝐷𝐻 + 𝐻+

Lactate ion Pyruvate ion iii. Hexocinase enzyme catalyses the inter conversion of glucose and ATP with glucose-6-

phosphate and ADP.

𝐴𝑇𝑃 + glucose𝒉𝒆𝒙𝒐𝒌𝒊𝒏𝒂𝒔𝒆↔ 𝐴𝐷𝑃 + glucose-6-phosphate

iv. Enolase enzyme catalyses a step of glycolysis, reversible dehydration of 2-phosphate glycerate to phosphenol phyruvate.

2-phosphoglycerate𝒆𝒏𝒐𝒍𝒂𝒔𝒆↔ phosphophenol pyruvate



v. Virtually every biochemical reaction is catalyzed by enzyme. So enzyme regulates life.

29. What are the environmental benefits of enzymes? Environmental benefits of enzymes are given below:

a. Replacing acids, alkalis or oxidizing agents in fabric desizing e.g., amylase. b. Use of enzymes in the tanneries to reduce the use of sulphides. c. Enzymes replace pumic stones for ‘stone washing’ of jeans. This reduces pumic waste. d. Enzymes used in animal feeds allow more complete digestion of feed leading to less animal

waste per pound gained. e. Replacing acids in the starch processing industry.

starch𝒂𝒎𝒚𝒍𝒂𝒔𝒆↔ glucose + arabinose

f. Using of enzymes in industry reduce industrial waste.

30. Discuss the role of enzyme as analytical reagent. Some enzymes are useful in the clinical laboratory for the measurement of substrates, drugs and

even the activities of other enzymes. The biochemical compounds (e.g. glucose, urea, uric acid, cholesterol) can be more accurately and specifically estimated by enzymatic procedures compared to the conventional chemical methods. A good example is the estimation of plasma glucose by glucose oxidase and peroxidase method.

31. Discuss the thermodynamics of enzymatic reaction. The enzyme catalyzed reactions may be broadly grouped into three types based on

thermodynamic (energy) considerations.

Isothermic reactions: The energy exchange between reactants and products is negligible e.g. glycogen phosphorylase.

Glycogen + 𝑃𝑖 → Glucose 1 − phosphate Exothermic (exergonic) reactions: Energy is liberated in these reactions e.g. urease.

Urea → 𝑁𝐻3 + 𝐶𝑂2 + energy Endothermic (endergonic) reactions: Energy is consumed in these reactions e.g. glucokinase.

Glucose + 𝐴𝑇𝑃 → Glucose 6 − phosphate + 𝐴𝐷𝑃

32. Short note: Coenzyme. Definition: Many enzymes catalyze reactions of their substrates only in the presence of specific

heat-stable, low molecular weight organic molecules called coenzyme. We get a factor or group without amino acid when we hydrolyze protein. This factor is called coenzyme. The coenzyme e.g. 𝐾+, 𝐹𝑒3+ etc. activates apoenzyme.

Coenzymes are heat-stable, dialyzable, non-protein organic molecules and the prosthetic groups of enzymes.

Classification: Coenzyme is classified as following:

1. Based on chemical characteristics: Coenzymes are classified into 3 groups e.g. a. Containing an aromatic hetero ring:

i. 𝐴𝑇𝑃 and its relatives, ii. 𝑁𝐴𝐷, 𝑁𝐴𝐷𝑃,

iii. 𝐹𝑀𝑁, 𝑇𝑃𝑃, 𝐵6 − 𝑃𝑂4.



b. Containing a non-aromatic hetero ring: i. Biotin,

ii. Lipoic acid. c. Containing no hetero ring:

i. Sugar phosphate, ii. Coenzyme 𝑄.

2. Based on functional characteristics: Coenzymes are classified into 2 groups e.g. a. Hydrogen transferring coenzymes:

i. 𝑁𝐴𝐷, 𝑁𝐴𝐷𝑃, ii. 𝐹𝐴𝐷, 𝐹𝑀𝑁,

iii. Coenzyme, iv. Lipoid acid.

b. Group transferring (other than hydrogen) coenzymes: i. 𝐴𝑇𝑃 and its relatives,

ii. 𝑇𝑃𝑃, iii. Coenzyme 𝐴, iv. Sugar phosphate, v. 𝐵6 − 𝑃𝑂4,

vi. Biotin and vii. Lipoic acid.

3. Based on nutritional characteristics: a. Containing vitamin 𝐵-complexes:

i. Coenzyme 𝐴, ii. 𝑇𝑃𝑃,

iii. 𝑁𝐴𝐷, 𝑁𝐴𝐷𝑃, iv. 𝐵6 − 𝑃𝑂4, v. Folic acid coenzyme,

vi. 𝐵12 coenzyme, vii. Biotin,

viii. FMN. Properties (characteristics):

Coenzymes are heat stable and non-protein organic molecules.

Coenzymes are dialyzable (non-colloid).

Coenzymes are derivatives of vitamin 𝐵-complex.

Coenzymes participate in: o Hydride (𝐻) and electron transfer reactions e.g. 𝑁𝐴𝐷, 𝑁𝐴𝐷𝑃, 𝐹𝑀𝑁, 𝐹𝐴𝐷. o Group transfer reactions e.g. coenzyme 𝐴, 𝑇𝑃𝑃, 𝐵6 − 𝑃𝑂4.

Functions: Coenzymes usually accept atoms or groups from a substance and transfer them to other

molecules. Coenzymes are less specific than enzymes and the same Coenzymes can act as such in a

number of different reactions. Coenzymes are also attached to the protein at a different but adjacent site, so as to be in a

position to accept atoms or groups that are removed from the substrate. Some Coenzymes performs specific function e.g.

NAD and NADP acts as hydrogen acceptor in dehydrogenation reactions. Coenzyme A carry acyl groups and they are used in the oxidative decarboxylation of

pyruvic acid and synthesis of fatty acids and acetylation.



TPP carry active aldehyde group. 𝐵6 − 𝑃𝑂4 involves in transamination reactions.

33. Coenzymes are considered as second substrate (or co-substrate) – explain.

Coenzymes are considered as second substrate for the following reasons:

Coenzyme involved reactions are of very great physiological significance. Example: in the anaerobic glycolysis, during muscular exercise, the reaction converting pyruvic acid into lactic acid causes reoxidation of 𝑁𝐴𝐷𝐻 to 𝑁𝐴𝐷. This 𝑁𝐴𝐷 then permits synthesis of 𝐴𝑇𝑃.

The chemical lchanges in the coenzyme exactly counterbalance those taking place in the substrate. Example: in oxidoreduction (dehydrogenase) reactions, one molecule of substrate is oxidized (dehydrogenated) and one molecule of coenzyme is reduced (hydrogenated).

Viva Special

Enzyme is protein in nature (exception – 𝑅𝑁𝐴 acting as ribozyme).

A for digit Enzyme Commission (EC) number is assigned to each enzyme representing the class (first digit), sub-class (second digit), sub-sub-class (third digit) and the individual enzyme (fourth digit).

Enzyme is 10,000 − 1,00,000 times faster or effective than catalyst.

Enzyme is used in biological detergent (Surf Excel).

Enzyme is used as bighting agent in detergents, cosmetics etc. (protein absorbs 280 𝑛𝑚 UV light.

Enzyme is used to test diabetic. Usual test cannot determine 𝛼 − 𝐷 − glucose. All 𝛼 − 𝐷 − glucose is first converted into 𝛽 − 𝐷 − glucose with the help of mutarotase. Then 𝛽 − 𝐷 − glucose is estimated by glucose oxidase.

Action of enzyme is very specific. There is only one chance of doing wrong in 1,00,00,000 times.

Urea is used to transform curly protein to linear protein structure. Urea makes bond with hydrogen, therefore, the protein is straightened down.

When it is vitamin is called coenzyme and when it is organic compound or metal is called cofactor or prosthetic group.

Apoenzyme + Coenzyme = Holoenzyme Protein part Non-protein part

As enzymes are made of protein they are non-dialyzable.

If the reaction is enzyme catalyzed or not can be differentiates by two tests: (i) heat sensitive test and (ii) acid test.

The name ligase comes from Greek ligate – to bind.

𝐾𝑚 = Michaelis-Menten constant or Brig’s and Haldane’s constant. It is defined as that substrate concentration which produces half of the maximum velocity. It is defined in terms of substrate concentration. Low 𝐾𝑚 is preferred because at low concentration we can achieve the maximum velocity.

Two categories of enzymes requires metals for their activity such as (i) metal-activated enzymes (not tightly held with metal) e.g. ATPase (𝑀𝑔++ , 𝐶𝑎++ ), enolase (𝑀𝑔++ ) and (ii) metalloenzymes (tightly held with metal) e.g. phenol oxidase (𝐶𝑢), pyruvate oxidase (𝑀𝑛), xanthine oxidase (𝑀𝑜), cytochrome oxidase (𝐹𝑒, 𝐶𝑢).

Suicide inhibition is a specialized form of irreversible inhibition where the original inhibitor is converted to a more potent form by the same enzyme that ought to be inhibited.

Specificity is a characteristic property of the active site.



The specificity of the enzyme is mostly dependent on the apoenzyme and not on the coenzyme.

Mechanism of enzyme action: o Lock and key model or Fischer’s template theory, o Induced fit theory or Koshland’s theory, o Substrate strain theory and o Michaelis-Menten theory.

Mechanism of enzyme catalysis: o Acid-base catalysis, o Substrate strain, o Covalent catalysis and o Entropy effects.

Regulation of enzyme activity in the living system: o Allosteric regulation, o Activation of latent enzymes, o Compartmentation of metabolic pathways, o Control of enzyme synthesis, o Enzyme degradation and o Isoenzymes.

The existence of life is unimaginable without the presence of enzymes – the biocatalysts.

Majority of the Coenzymes (𝑇𝑃𝑃,𝑁𝐴𝐷+, 𝐹𝐴𝐷, 𝐶𝑜𝐴) are derived from 𝐵-complex vitamins in which form the later exert their biochemical functions.

Competitive inhibitors of certain enzymes are of great biological significance. Allopurinol, employed in the treatment of gout, inhibits xanthine oxidase to reduce the formation of uric acid. The other include aminopterin used in the treatment of cancers, sulfanilamide as antibactericidal agent and dicumarol as an anticoagulant.

Enzyme inhibitors exert their effect by acting on the apoenzyme, coenzyme, prosthetic group or activators present in the enzyme system or by interfering with the binding of substrate to the enzyme.

Feedback (or end product) inhibition is a specialized form of allosteric inhibition that controls several metabolic pathways e.g. 𝐶𝑇𝑃 inhibits aspartate transcarbamolyase; cholesterol inhibits 𝐻𝑀𝐺 coenzyme 𝐴 reductase. The end product inhibition is utmost important to cellular economy since a compound is synthesized only when required.

Allosteric site is the site other than active site which regulates the activity of the enzyme. It binds with other than the substrate. The compound that binds with it is known as allosteric effector. A modifier may binds with it.

The nerve gas (diisopropyl fluorophosphates), first developed by Germans during Second World War, inhibits acetylcholine esterase, the enzyme essential for nerve conduction and paralyses the vital body functions. Many organophosphorus insecticides (e.g. melathion) also block the activity of acetylcholine esterase.

Penicillin antibiotics irreversibly inhibit serine containing enzymes of bacterial cell wall synthesis.

Certain 𝑅𝑁𝐴 molecules (ribozymes) function as non-protein enzymes. It is beloved that ribozymes were functioning as biocatalysts before the occurrence of protein enzymes during evolution.

Isoenzymes are the multiple forms of an enzyme catalyzing the same reaction which however, differ in their physical and chemical properties. 𝐿𝐷𝐻 has five isoenzymes while 𝐶𝑃𝐾 has three.

Turnover number: The combined processes of enzyme synthesis and degradation constitute enzyme turnover.

Process of enzyme isolation:



o Dialysis, o Absorption, o Differential heat or 𝑝𝐻 denaturation, o Differential centrifugation, o Gel filtration, o Electrophoresis and o Precipitation with varying salt concentration.

Units of enzyme activity: o 𝐾𝑎𝑡𝑎𝑙: One 𝑘𝑎𝑡 denotes the conversion of one mole substrate per second (𝑚𝑜𝑙/𝑠𝑐𝑒).

Also 𝑚𝑘𝑎𝑡, 𝜇𝑘𝑎𝑡 and so on. o International unit (𝐼𝑈): One 𝐼𝑈 denotes the amount of enzyme activity that catalyses

the conversion of one micromol (𝜇𝑚𝑜𝑙) of substrate per minute. o 1 𝐼𝑈 = 60 𝜇𝑘𝑎𝑡 o 1 𝑛𝑘𝑎𝑡 = 1.67 𝐼𝑈

Coenzyme 𝐴: It is composed of 𝐴𝑇𝑃, pantothenic acid and 𝛽-mercaptoethylamine. So it is the coenzyme form of pantothenic acid.

Coenzyme 𝑄: Coenzyme 𝑄 is a derivative of benzoquinone in which one of the substitute is polyisoprenoid chain. It is also known as ubiquinone.

Subdivision of cofactor:

Cofactors

Organic (coenzymes)

Tightly bound (prosthetic

group)

Loosely bound(second

substrate)

Inorganic (metal ions)

Tightly bound (metalloenzyme)

Loosely bound (ion activators)

notes on enzyme

Science