Chapter five


An enzyme is a biological molecule that catalyzes biochemical reaction under mild physiological conditions, which increase the rate of reaction by lowering the activation energy without any change in the over all process. Enzymes are produced by the living organism and are present in very small amounts in various cells. Almost all the functions of the body such as digestion, breathing, synthesis, breakdown of carbohydrates, fats, and proteins are catalyzed and controlled by specific enzymes.

The substance (molecule) upon which an enzyme acts is called the Substrate [S] and Enzyme will convert the Substrate into EnzymeSubstrate complex (E-S) then to product [P].

The complete structure of apo enzyme and prosthetic group is called holo enzyme. Holo enzyme = Protein part + Non protein part Apo enzyme Prosthetic group(Cofactor) .Protein nature of enzymes: All enzymes are proteins with the exception of a small group of RNA molecules. like ribozymes. When the enzyme is a conjugated protein. the protein part is called as apo enzyme and the non protein part is called as the prosthetic group or cofactor. Enzymes are either simple or conjugated proteins.

g. .Some enzymes have only one polypeptide chain in their structure is called monomeric enzyme. Ribonuclease and pepsin. e. Like lactate dehydrogenase have four polypeptide chains. Many other enzymes have more than one polypeptide chains in each molecule are called oligomeric enzymes.

but required only in very small amounts.Properties of enzymes: 1.All enzymes are proteins with the exception of a small group of RNA molecules. 4.They have enormous power for catalysis. . 3. 2. ribozymes.Enzymes are highly specific for their substrate.They accelerate the reaction.

Enzymes lower activation energy. 8. .5. 7.Some enzymes are regulatory in function. 6.Enzymes possess active sites at which interaction with substrate take place.They form substrate complex as intermediates during their action.

Intracellularly 2.Extracellularly .Localization of enzymes: Enzymes are localized either: 1.

Krebs cycle enzymes….. e.. glycolysis enzymes.g. .Intracellularly Many enzymes usually act within the cells in which they are produced and hence are called intracellular enzymes or endoenzymes.

pepsin.Extracellularly Certain enzymes are secreted out from the cell and catalyze reactions outside the cell origin.g. trypsin. lipase. E. digestive enzymes: αamylase. .. and hence are known as extracellular enzymes or exoenzymes.

Enzyme once dissociated from the complex is free to combine with another molecule of substrate and form product in a similarly way.Mechanism of enzyme action: The substrate [S] binds to enzyme[E] on a specific sites called as the active sites to form enzymesubstrate complex [E-S]. The active site (or catalytic site) is made up of several amino acids. . which then E-S complex converted to enzyme-product complex [E-P] which further dissociated to form product [P] and [E] back.

Active site:
It is a place on the enzyme where combined with the substrate and made up of amino acids

Models of enzyme-substrate complex formation:
Two models for substrate binding to the active site of the enzyme have been proposed to explain the specificity that an enzyme has for its substrate:

1- Lock and key models (Rigid template model or Emil Fisher):
This model assumes that the active site of an enzyme is rigid or inflexible (the lock) and the substrate (the key) fits into a specific rigid (the active site) on the enzyme to form the enzymesubstrate complex.


2. The interaction between enzyme and substrate causes to change in the shape of the active site of enzyme to form the enzyme-substrate complex.Induced fit model (Koshland model): The lock and Key hypothesis does not explain the broad specificity of some enzymes. Also the molecular shape of active sites is not always complementary to that of the substrate. This model assumes that the active site of an enzyme is flexible. The induced fit attempt to over come this difficulties. .

Prentice Hall © 2007 Chapter Nineteen 17 .The induced-fit model model of enzyme action has a flexible active site that changes shape to best fit the substrate and catalyze the reaction.


(a) The polypeptide enters the active site (b) Hydrogen transfer allows formation of a strained intermediate (c) The peptide bond is broken.Hydrolysis of a peptide bond by chymotrypsin. Prentice Hall © 2007 Chapter Nineteen 19 .

and chymotrypsin. . . the common name of an enzyme often tells us a great deal about the function of an enzyme. pepsin. the names of the enzymes and the reactions that they catalyze must simply be memorized. as in ureasewhich catalyze the hydrolysis of urea and lactase catalyzes the hydrolysis of the disaccharide lactose. In these cases. trypsin. enzymes often were named by adding the suffix-ase to the name of the substrate upon which they acted.Enzyme Nomenclature: Traditionally. A few examples include catalase. Yet other enzymes have historical names that have no relationship to either the substrates or the reactions that they catalyze.

The International Union of Biochemistry and Molecular Biology (IUBMB) has established a system whereby all enzymes are classified into six major classes. the type of reaction that is catalyzed. These systematic names tell us the substrate. and the name of coenzyme involved in the reaction. each subdivided into subclasses that are further subdivided. . For example. alcohol dehydrogenase is alcohol: NAD+ oxidoreductase because it catalyzes an oxidation–reduction reaction and the electron donor is an alcohol and the acceptor is NAD+.

this number called: Enzyme commission numerical code (EC) and it contains 4 digits: .In addition to naming enzymes. the IUBMB classifies the enzymes by giving each enzyme a number.

-CHO. -OH. FAD. Second: indicates the functional group upon which the enzyme acts e. Fourth: It indicates the serial number of enzyme.First: indicates the class of the enzyme (one of the six).g. NAD.g. Third: indicates the acceptor e. .

For example: Lactate dehydrogenase: Alcohol Dehydrogenase .

A according to the IUBMB system. Enzymes are classified into six major classes as follows: .

1-Oxidoreductase: Enzymes. . which catalyze the transfer of hydrogen or addition of oxygen. e.g.. lactate dehydrogenase or catalase.


They catalyze the transfer of some groups (as methyl , formyl ,carboxyl ,nitrogenous, phosphorus & sulfur containing groups except hydrogen) from one molecule to another molecule . Like amino transferase or hexokinase.

3- Hydrolase:
Enzymes that catalyze the cleavage of C-O, C-N, C-C, and some other bonds with the addition of water. Like: all digestive enzymes like αamylase, pepsin, trypsin,…


. Like: pyruvate decarboxylase or aldolase.4.Lyases: Lyases catalyze the cleavage of C-O. C-C bonds without adding water. C-N.


Like phosphoglycerate mutase. isomerase. and epimerase. glucose-6-phosphate mutase.5-Isomerase: Isomerase rearrange the functional groups within a molecule and catalyze the conversion of one isomer into another. .


6.Ligases (Synthetase): These enzymes link two substrates together with the hydrolysis of ATP to ADP and Pi. They may form or broken of C-O. C-C. and C-S.…. C-N. . Like glutamine synthetase or peptide synthetase. and pyruvate carboxylase.







There are three types of specificity: .Specificity of enzyme action: Enzyme specificity is the ability of an enzyme to bind on one or few substrates and catalyze a reaction.

N-methyl urea and thiourea are not hydrolyzed by ureaes.. e. .Absolute specificity: Some enzymes will act only on one substrate and catalyze one reaction.g. the enzyme urease catalyzes the hydrolysis of urea: The catalysis does not take place when the structure of urea is changed. For example.1.

g.. . and lipase.2. peptidase. e. hexkinase.Group specificity (non-absolute specificity): These enzymes can catalyze a group of structurally similar compounds.

Human enzymes are specific for L-amino acid and Dcarbohydrates and the enzymes involved in digestion and metabolism recognize only those particular stereo isomers.Stereo specificity: a. .3.Optical specificity: These enzymes are capable of acting only with one form of two optical isomers L or D.

. For example: alanine racemase. interconvert the two optical isomers of a compound.And some enzymes.

Geometrical specificity: Some enzyme react with one form . trans fumaric acid malic acid .b.the cis or trans. For example: Fumarase catalyze the interconversion of trans fumaric acid and malic acid.

Commonly cofactors may be metal ions such as Zn+2. enzymes do not show biologic activity. In the absence of the cofactor. this organic molecules which are many enzymes needed their activity called coenzyme.… or may be organic molecules. and almost coenzymes are derivatives of vitamins. NADPH. Fe+2.Cofactors and Coenzymes: Some enzymes require an additional non protein part called cofactor that is needed for enzyme activity. . and FADH. Like NADH.




Enzyme Cofactors 53 .

• Zn2+.Metal Ions as Cofactors • Many active enzymes require a metal ion. a cofactor for carboxypeptidase. stabilizes the carbonyl oxygen during the hydrolysis of a peptide bond. 54 .

For example: .Isoenzymes or isozymes: Isoenzymes are multiform (isomers) of the same enzyme that catalyze the same reaction but differ in structure and in the kinetic constant Km and Vmax.

where as in skeletal muscle the M4 form predominant. H3M (LDH2). made up of two polypeptide M (Muscle) type and H (Heart) type. LDH have five isoenzymes: H4 (LDH1).Lactate dehydrogenase (LDH) which is catalyze the reversible oxidation of lactate to pyruvate. and M4 (LDH5). HM3 LDH4). In the heart tissue H4 is the dominant form. . H2M2 (LDH3). Since LDH is a tetramer.

Isoenzymes H subunit Isoenzymes of lactate dehydrogenase M subunit .

which may be either M (Muscle) type or B (Brain) generating three isoenzymes CK1 (BB).Creatine kinase are dimmer. in muscle 85% of the enzyme is in MM form and 15% in the MB form in the myocardium. CK3 (MM). that are made up of two types of polypeptide chains. BB form predominates in brain. . CK2 (BM).

which must be cleaved (a part of the polypeptide or a few amino acid residue) to be activated.Zymogene (proenzymes): Some enzymes are synthesized in an inactive forms called zymogene or proenzymes. For example: .


The secretion of the enzyme in proenzyme (inactive form) to: 1.Prevents auto digestion of tissues 2.Prevents intravascular coagulation of blood .



the conditions of assay must be specified). .Enzyme unit: In many situations. The International Commission on Enzymes defines One International Unit of enzyme as the amount that catalyzes the formation of one micromole of product in one minute(Because enzymes are very sensitive to factors such as pH.U(International Units). temperature. one katal= 6 x 107 I. and ionic strength. the actual molar amount of the enzyme is not known. Thus. One katal is that amount of enzyme catalyzing the conversion of one mole of substrate to product in one second. However. its amount can be expressed in terms of the activity observed. Another definition for units of enzyme activity is the katal.

Or one IU =16. has recently been introduced. One katal = 6 x 107 international units.U. One katal (kat) indicates the amount of enzyme for the transformation of 1 mole of substrate per second.U. An enzyme specific activity.Enzyme activity assay and unit of enzyme activity Enzyme activity is measured in international units (I. a quantity that is used to monitor enzyme purification. is defined as the number of international units per milligram of protein. is defined as the amount of enzyme that produces 1μmol of product per minute. A new unit for measuring enzyme activity called the katal.) One I.67×10-9 kat .

3-Enzyme concentration 4-Substrate concentration 5.Inhibitor.PH.Factors affecting the velocity of enzyme reaction: Various factors that affect enzyme activity are: 1.Temperature. . 2.

O. The temperature at which maximum amount of substrate is converted to the product per unit called the optimum temperature (O. .T is that temperature at which the activity of the enzyme is the maximum. The rate reach a maximum and then falls (or begins to decrease).1-Temperature: Increase in temperature increases the rate of enzyme catalyzed reactions. The decrease in velocity (rate) at higher temperature is due to denaturation of enzymes and the enzyme become inactive. Most of the body enzymes have the O. .T close to 3545OC.T).


PH of most enzymes are in the range of 4-9. The velocity is increase when PH is increased. arginase 8. Above the O. the enzyme activity at this maximum PH is called maximum PH or optimum PH (O. alkaline phosphate =10-11. . The O.9-9.PH is that PH at which the enzyme activity is the maximum.2. reaches a maximum. O.PH).9.Effect of PH: Velocity of an enzyme catalyzed reaction is dependent upon PH. Whilesome enzymes like pepsin PH=2.PH the velocity of reaction decrease and the enzyme are denaturated.



Enzyme concentration: The velocity of enzyme catalyzed reaction is directly proportional to the concentration of enzyme. the rate of reaction is increased to. .3. When the concentration of enzyme is increase. when the substrate concentration is in large excess in the medium.

the initial velocity of reaction increases proportionally with the increase in substrate concentration. the velocity of reaction remains unchanged however. . At high Substrate concentration. This is called as maximum velocity (Vmax).Substrate concentration: At a low concentrations of substrate. Further increase in Substrate concentration. The Vmax is the velocity of the reaction at high Substrate concentration when all active site of the enzyme are filled with the substrate. the velocity of the reaction increase less.4. much the Substrate concentration is increased.


has no effect on the velocity and Vmax is attained. so that any further increase in [S]. Here the velocity will be proportional to [S].The reason of these that at low Substrate concentrations. . active site of the enzyme will be in the uncombined form E. The maximum velocity all the enzyme is saturated with substrate and present as the E-S complex.

50% [S] or Km C. High. Low [S] B. saturating [S] .Substrate Saturation of an Enzyme A.

low Km indicates higher affinity of the enzyme for the substrate and vice versa. .The Substrate concentration [S] where the velocity of the reaction V0 is one half of the maximum velocity (1/2 Vmax) is called Michael constant Km. Km indicates the affecting of the enzyme for the substrate .

The relationship between reaction rate and Substrate concentration is described mathematically by the Michaels –Menton equation as follows: .

A more accurate method of determining values for Vmax and Km uses line weaverBurk equation by taking the reciprocal of the Michaels –Menton equation: .Some times at higher Substrate concentration at maximum velocity. 1/2Vmax and Km may be difficult to determine.

Where the intercept on the Xaxis is equal to -1/Km and the intercept on the Y-axis is 1/Vmax. a straight line is obtained.Where 1/Vo is plotted against 1/[S]. .

The LineweaverBurk doublereciprocal plot.Linear Plots Can Be Derived from the MichaelisMenten Equation The LineweaverBurk equation. .

These compounds include: drugs.5. and natural products of the enzyme reaction .Inhibitors: Inhibitors are chemical compounds that bind to the enzyme which decrease or stop the activity of an enzyme. antibiotics. toxins.


Un Competitive inhibition .Competitive inhibition 2.Non Competitive inhibition 3.Inhibitors are classified based on whether they bind to active site or any other side (rather than the active site) on the enzymes: 1.

E-S complex is not formed. Hence the rates of the reaction will decrease. so inhibitor [I] competes with the substrate for the active site. E-I can not be converting to product. it pushes the inhibitor from the active site and combines with the enzyme to form E-S complex which can be converted to product. Therefore. Thus by increasing the substrate concentration [S]. instead E-I complex formation take place. .a-Competitive inhibitions: The inhibitor is a structural similar to the substrate.


Like Inhibition of the enzyme succinate dehydrogenase by malonate . can be combined with the succinate dehydrogenase instead of succinate. Malonate. The inhibition can be removed by increasing the concentration of succinate. .

The effect of enzyme inhibition Succinate Fumarate + 2H++ 2e- Succinate dehydrogenase CH2COOH CH2 CH2COOH COOH Malonate © 2008 Paul Billiet ODWS COOH CHCOOH CHCOOH .

Sulfa Drug Is Competitive Inhibitor Domagk (1939) Para-aminobenzoic acid (PABA) Bacteria needs PABA for the biosynthesis of folic acid H2NPrecursor -COOH Folic acid Tetrahydrofolic acid H2N- -SONH2 Sulfa drugs has similar structure with PABA. and inhibit bacteria growth.197 . Sulfanilamide Sulfa drug (anti-inflammation) Adapted from Bohinski (1987) Modern Concepts in Biochemistry (5e) p.

. The effect of inhibitor can be removed by increasing the concentration of the substrate. Enzyme affinity of the substrate is decrease.Km is increased but Vmax is constant.

.Non competitive inhibition: In this type of inhibition no competition occurs between substrate and inhibitor. Inhibitor is usually structurally different from the substrate and the inhibitor binds with the enzyme at a site (place) other than the active site. forming E-I and ESI complexes. The inhibitor may combine with E and ES complex.2.


This is an enzyme of the electron transport chain and generates ATP. Cu and Fe of the enzyme cytochrome oxidase. These can be removed by using a chelating agent such as EDTA .Like cyanide which are formed complexes with metal ions.


The effect of inhibitor can not be removed by increasing the concentration of the substrate. .Km is constant and Vmax is decrease.

Inhibitor binds to enzyme – substrate complex.Uncompetitive inhibition: Here inhibitor does not have any affinity for free enzyme.3. but not to the free enzyme .

Like inhibition of placental alkaline phosphatase by phenyl alanine. .

. The effect of inhibitor can not be removed by increasing the concentration of the substrate.Both Km and Vmax are decreased.

Enzymes are the tools of life. .