Enzymes are biocatalysts that accelerate biochemical reactions without being consumed. They are typically proteins that specifically bind to substrates and lower their activation energy through transition states, speeding up reactions. Enzymes can be intracellular or extracellular, and require cofactors, metal ions, or allosteric regulation for optimal activity. Their activity is affected by factors like pH, temperature, substrate and inhibitor concentration. Enzymes are classified by the type of reaction they catalyze and can exist as isozymes or allosteric variants that perform the same function. Inhibition can be reversible via competitive or non-competitive binding, or irreversible through covalent modification of the active site.

1. ENZYMES

2. WHAT ARE THEY ???
 Biocatalysts – catalyse &enhance the rates of biochemical reactions.
 Property of accelerating certain chemical reactions.
 Without themselves doesn’t undergo any change.
 Not permanently changed in the process.
 Are specific in action.
 Most enzymes are proteins in nature.
 They are thermo labile.

3. RELATED TERMS
 Intracellular Enzymes –Enzymes produced by the cells of a particular tissue &
function within the cell.
 Eg ; Enzymes of TCA cycle, Glycolysis etc.
 Extracellular Enzymes – Enzymes produced by the cells from where they are
liberated to use in other tissues.
 Eg : Proteolytic enzymes of GIT.
 Oligomeric Enzymes - Enzymes with two or more subunits.
 Multienzyme complex – Enzymes occur in a single complex form. Eg, Pyuruvate
dehydrogenase.
 Zymogen (Proenzymes) – No.of proteolytic enzymes found in the digestive tract or
in the blood are found in an inactive form. Eg; Chymotrypsin.
 Holoenzymes – Enzymes that require the presence of certain additional nonprotein
component for its optimum activity.
 Cofactors – the additional part, organic (coenzymes) or inorganic (activators).
 Apoenzymes – Enzymes without it’s cofactor.

4.  Coenzymes –
1. thermostable
2. low molecular weight
3. considered as second substrate
 Eg ; thiamine pyrophosphate
 Several apoenzymes require the presence of metal ions – Mg 2+ ,Zn2+ etc.

7. STRUCTURE OF ENZYMES
 Enzyme binds with is specific substrate – Enzyme –Substrate
complex.
 It binds at a site called Active site.
 It is a small cleft in an enzyme where the chemical groups are
arranged in an order.
 At the end of the reaction the substrate is converted into the
product.
 Enzyme remains unchanged.
E + S E-S E + P

8. HOW ENZYME WORKS
 Energy barrier separating the reactants & products in chemical reactions – Free
energy of activation.
 It is the difference between the energy of reactant & high energy
intermediates that occur during the formation of a product.
 Peak of free energy activation – transition state – high energy intermediates are
formed.
 Enzyme lowers the energy required for the activation to the transition state.
 Without a catalyst, the reaction will occur only if heat energy is added to the
system.
 With an enzyme, reaction may proceed at the normal temperature.

10. MECHANISM OF ENZYME ACTION
 Interaction of enzyme with substrate can be explained by two models ;
1) LOCK & KEY MODEL or RIGID TEMPLATE or EMIL FISCHER
2) INDUCED FIT MODEL or HAND –IN GLOVE or DANIEL E
KOSHLAND

11. LOCK & KEY MODEL
 In this model,
1. Enzyme is preshaped
2. Active site has a rigid structure complementary to that of the substrate.
3. Substrate fit into the active site like the way that a key fits into a lock.
 Some enzymes can bind only a specific substrate but will not bind another
compound.

12. INDUCED FIT MODEL
 In this model;
1. The enzymes are flexible
2. Shapes of the active site can be modified by the binding site of the substrate.
3. Substrate induces a conformational changes in the enzyme, in the same way
placing a hand into the glove induces the change in the shape of the glove’s
shape.
4. Conformational change in enzyme in turn alters the orientation &
configuration of the bound substrate.
 This model is more accurate.
 Also explains action of allosteric modulators & competitive inhibition on
enzymes.

14. Units of enzyme activity
 Are never expressed in terms of their concentration ,but as their activities.
 They are:
1. Katal –maintain uniformity in the expression of enzyme activities.
a) One kat denotes the conversion of one mole substrate per second.
b) Also expressed as millikatals (mkat), microkatals (µkat).
2. International Units (IU)
a. Amount of enzyme activity that catalyses the conversion of one micromol of
substrate per minute.
b. 1 IU =60 µkatal

15. CLASSIFICATION
 Acco.to IUB classification enzyme is characterized by a code number –
Enzyme code number or ‘EC’ number.
 Classified into six major classes:
1. EC -1 : Oxidoreductases
2. EC -2 : Transferases
3. EC -3 : Hydrolases
4. EC -4 : Lyases
5. EC -5 : Isomerases
6. EC -6 : Ligases
MNEMONIC : OT-HILL

16. EC -1 : Oxidoreductases
 Enzymes that catalyze oxidation-reduction reaction.
 Include ;
I. Dehydrogenases
II. Reductases
III. Oxidases
IV. Peroxidases

17. EC -2 : Transferases
 Enzymes that catalyze the transfer of a functional group –carboxyl, amino,
methyl or phosphoryl from one molecule to another.
 Include ;
I. Amino transferase or transaminase
II. Kinase
III. Transcarboxylase

18. EC -3 : HYdrolases
 Enzyme that catalyze the cleavage of C-O, C-N, C-C & some other bonds with
the addition of water.
 Includes:
I. Acid phosphatase
II. Digestive enzymes like pepsin, trypsin &chymotrypsin

19. EC -4 : Lyases
 Enzyme that catalyzes the cleavage of C-O, C-C & C-N bonds by means other
than hydrolysis & oxidation.
 Give rise to compounds with double bonds or catalyze the reaction ,by the
addition of group to a double bond.
 Includes;
I. Aldolase
II. Arginosuccinase

20. EC -5 : isomerases
 Enzyme catalyses intramolecular structural rearrangement in a molecule.
 Also called epimerases or mutases.
 Includes;
I. Triphosphate isomerase
II. Phosphohexose isomerase

21. EC -6 : Ligases
 Enzyme catalyzes the joining of two molecules coupled with hydrolysis of ATP.
 Includes ;
I. DNA ligases
II. Pyruvate carboxylase

22. Enzyme kinetics
Michaelis – Menten equation
 Occurs in two stages;

24. TYPES OF SPECIFICITY
.
Specificit
y
Substrate
specificit
y
Absolute
Specificit
y
Relative
substrate
Specificit
y
Reaction
Specificit
y
Stereo
specificit
y

28. Factors affecting the enzyme activity
1. pH -
 Enzyme has optimum pH at which the activity of enzyme is maximum.
 ↑ or ↓ pH causes ↓ in enzyme activity.
2. Temperature -
 Enzyme activity is more at optimum temperature.
 Strong change in this results in loss of enzyme activity.
 Optimum temp.of enzymes in human body - 37˚c
3. Concentration of substrate –
 At low substrate conc.enzyme molecules are free initially.
 ES complex formation is proportional to substrate concentration
 ↑ conc. Enzyme molec. are saturated with substrate
 No change in further

29. 4. Concentration of enzyme –
 Velocity of enzyme reaction is directly proportional to the enzyme
concentration
5. Time :
 Under optimum condition of temperature & pH, the time required for enzyme
reaction is less.
6. Effect of Inhibitors :
 Presence of inhibitors ↓ the rate of the enzyme reaction.
7. Effect of physical agents :
 Such as light rays accelerate or inhibit enzyme reactions.
8. Effect of product :
 Accumulation of products of the reaction causes the inhibition of enzyme
activity.
 Limit the rate of formation of product.

30. 9. Effect of activators & coenzymes:
 Activity of main enzymes depend upon certain metallic cations like Mg2+, Cu2+,
Mn2+, Na2+, Ca2+ etc. for their optimum activity.
 Absence of activators and coenzymes make the enzymes functionally inactive.

32. Isoenzymes
 Also called isozymes
 Are multiple forms that catalyze the same biochemical reaction
 Different forms of a single enzyme.
 Same catalytic activity, differ structurally, physically & chemically.
 Eg:
A. Lactate dehydrogenase – Five isoenzymes (LDH1, LDH2, LDH3, LDH4 &LDH5)
B. Creatine kinase – three isoenzymes (CK1, CK2, CK3 )

34. Allosteric Enzymes
 Are multi subunit enzymes with a separate allosteric site in addition to the
catalytic/active site.
 Is a regulatory enzyme.
 Allo means ‘other’ & steros means ‘space or site’-those having other sites.
 They also have one more site for binding regulatory metabolites – Modulators.
 Eg ; phosphofructokinase –I, pyruvate dehydrogenase, Isocitrate dehydrogenase.

35. Enzyme inhibition
 Defined as the substance which binds with the enzyme & brings about a
decrease in catalytic activity of that enzyme.
 Divided into :
Enzyme
Inhibitors
Reversible
Competitive
Non-
competitive
Irreversible
Substrate
analogue
Group
specific
Suicide or
mech.based
inactivation

36. Reversible Inhibition
 Inhibitor binds non-covalently with enzyme.
 Enzyme inhibition is reversed, if the inhibitor is removed.
 Divided into :
A. Competitive Inhibition:
 Inhibitor (I) closely resembles the substrate – Substrate Analogue.
 Inhibitor competes with the substrate for the active site of the enzyme &
binds.
 But does not undergo catalysis.
 This inhibition could be overcome by a high substrate concentration.
 Km value ↑ & Vmax remains unchanged.
 Eg: Succinate dehydrogenase with succinic acid as its substrate. Malonic
acid has similar structural similarity with succinic acid and compete.

37.  Antimetabolites: chemical compounds that block the metabolic reactions by their
inhibitory action on enzymes.
B. Non –competitive Inhibition :
 Inhibitor binds at a site other than the active site on the enzyme surface.
 Impairs the enzyme function.
 Has no structural resemblance with substrate.
 Does not interfere with the enzyme-substrate binding.
 Catalysis is prevented, due to change in enzyme conformation.
 Generally, binds with enzyme as well as ES complex.
 Km value is unchanged, Vm is ↓.
 Eg ; heavy metal ions (Ag+, Pb+, Hg2+ etc.),inhibits enzyme binding with
cysteinyl sulfhydryl groups.

39. Irreversible inhibition
 Inhibitor binds covalently with an enzyme & forms a stable complex.
 Cannot be released by dilution or increasing the effect of substrate concentration.
 No effect on Km, ↓ Vm.
 Eg: Iodoacetate is an irreversible inhibitor of the enzyme papain & glyceraldehyde
3-phosphate dehydrogenase.
 Di-isopropylphosphoflouride –nerve gas binds with enzyme containing serine at the
active site.
 Divided into ;
A. Substrate Analogue or affinity labels ;
 Structurally similar to the substrate.
 Posses a highly reactive group
 Covalently reacts with active site & permanently block the site.
 Eg; 3-bromoacetol phosphate (BAP).

40. B. Group specific:
 Inhibitors react with specific R-groups (side chains) of amino acid residues in the
active site.
C. Suicide Inhibitor or Mechanism based inactivation ;
 Unreactive until they bind to the active site of an enzyme.
 Original inhibitor is converted to more potent by the same enzyme which is going
to get inhibited.
 Act as drugs for example
 Also called mechanism based inactivation – because they utilize the normal
enzyme reaction mech.to inactivate the enzyme.
 Eg: Allopurinol, an inhibitor of xanthine oxidase gets converted to alloxanthine

41. Clinical significance of enzymes
 Certain enzymes are used :
1. For the diagnosis of the disease
2. As therapeutic agents
3. As analytical agents.
 DIAGNOSTIC USE OF ENZYME :
1. Marker of cellular damage
2. Measurement of enzyme in plasma is used in the investigation of liver, heart,
muscle & pancreas diseases

43.  As therapeutic agents:
1. Some enzymes are used in the treatment of some diseases.
2. Eg;
a. Collagenase : used for debridement of dermal ulcers & burns
b. Fibrinolysin: used in the venous thrombosis & artery embolism (blood clot).
c. Lysozyme : found in human tears &egg white, used in the infection of eye.
 Analytical use of enzymes:
1. Can be used as reagents & labels.
2. Determining the serum concentration of drugs, hormones.
3. Commonly used label enzymes are:
 Alkaline phosphatase
 Peroxidase