Undergraduate Enzymology for MBBS students

1. ENZYMOLOGY
Dr. Aritri Bir
Associate Professor
Dept. of Biochemistry
IQ City Medical College, Durgapur.

2. Enzymology (part I)
• What is ENZYME ?
• Nomenclature of ENZYMES.
• Classifications of ENZYMES.
• Structural organization of ENZYME.
• What are Holoenzyme, Apoenzyme, Prosthetic group, Metallozyme,
Metal dependent enzyme, coenzyme ?
• Mechanism of enzyme action.
• Factors affecting enzyme kinetics.

3. Introduction
 AII enzymes are proteins
except – RNA molecules
 Organization of Enzyme molecule :
 Monomeric and multimeric enzymes
 Multifunctional enzyme
 Multienzyme complex
 The catalytic activity of enzyme depends on the integrity of its native
conformation. - Explain
 The catalytic activity of enzyme is usually lost if
 If an enzyme is denatured or dissociated into its subunits
 If an enzyme is broken down into its component amino acids
 Thus the primary, secondary, tertiary, and quaternary structures of protein
enzymes are essential to their catalytic activity.

4. Introduction - terminologies
• Proenzyme / Zymogens :
• Cofactors –
Additional non-protein molecule essential for catalytic activity.
• Two types : 1. Metal ions 2. Coenzymes
• Coenzymes : Non-protein organic molecules.
• Prosthetic groups : conenz or cofactors bound covalently to enz.
• Apoenzymes & Holoenzymes :

5. Metal Activated Enzymes versus Metalloenzymes
Metal-activated enzymes : shows greater catalytic activity by the
presence of a mono or divalent metal ion exterior to the protein (in the
assay medium).
• Metal-activated enzymes typically lose activity during purification.
• Hexokinase requires Mg2+
Metalloenzymes - metal as cofactor bound firmly to a specific region
on the protein surface.
• Metal ions cannot be separated without molecular breakdown of enzyme.
• Cytochrome oxidase contains Fe3+ and Cu2+
• Catalase contains Fe2+

6. IUBMB Classification
Enzymes are classified according to the type of reaction they catalyze.
Cl. no Class Name Reaction they catalyze
1 OXIDOREDUCTASE Transfer of electrons (Oxidation & Reduction)
2 TRANSFERASE Transfer of groups
3 HYDROLASE Hydrolysis (cleavage of substrate by adding water)
4 LYASE Addition of groups to double bonds, or formation of double
bonds by removal of group
5 ISOMERASE Transfer of groups within molecules to yield isomeric forms
6 LIGASE Formation of C-C, C-S, C-O, AND C-N bonds by condensation
reactions coupled to cleavage of ATP or similar cofactor

7. Nomenclature of ENZYMES
• Hexokinase – IUBMB no. 2. 7. 1. 1.
Class 2 Subclass 7
– transfers
phosphate
group
1st sub-subclass
in which the
acceptor of
phosphate
group is alcohol
First enzyme of the
1st sub-subclass

8. ENZYME KINETICS
• Study of the rate of enzyme-catalyzed reaction and the factors affecting it.
• Kinetic theory of any chemical reaction ???
• Energy Barrier & Activation energy
• Transition state
• How the enzymes work??
• ES complex formation (reversible) by weak non-covalent interaction.
• Each interaction release small amount of free energy
• Binding energy released used to lower the activation energy
• Reduces entropy
• Desolvation of substrate
• Proper alignment of functional group

9. FACTORS affecting THE RATE OF ENZYME
CATALYSED REACTION
• Temperature
Each enzyme works in an optimal (narrow range) temperature – explain.
• Initial temp increase → Activity increases gradually
• At optimal temp → Activity maximum
• Further increase in temp → Activity lost
• pH
• Enzyme concentration
• Substrate concentration

10. Enzymology (part II)
1. Measurement of Enzyme activity.
2. Enzyme specificity
3. Michaelis Menten EQUATION.
4. Exception of MM Equation
5. Define Km and Vmax. What is its importance?
6. Determination of Km and Vmax by double reciprocal plot.
7. Turnover number of enzyme.
8. What are the different mechanism of enzyme catalysis?

11. Measurement of Enzyme Activity
• Enzymes are quantitatively expressed in terms of catalytic activity.
• The catalytic activity of an enzyme is indicated by the conversion of
substrate into product.
• International Unit:
Amount of enzyme catalyzing the transformation of one micromole
of substrate into product in one minute in 250 C.
• KATAL:
The amount of enzyme which converts 1 mole of substrate into
product in 1 second.
1 Katal = 6 x 107 IU

12. Specificity of the enzyme
The enzyme specificity is of Three different types:
1. Optical specificity – for substrate and Product.
2. Reaction Specificity – one enzyme catalyzes only one
of the various reactions.
3. Substrate specificity
a. Absolute
b. Relative
i. Group dependent
ii. Bond dependent

13. Michaelis Menten Equation
• The relation between Substrate conc and Reaction velocity is
explained by the equation.
Vo =
Vo = initial rate of reaction
Vmax = maximum rate of reaction
Km = MM constant
[S] = Substrate conc.
Vmax [S]
Km + [S]

14. Michaelis Menten Equation
The prerequisite or assumptions:
• Substrate concentration is much higher than the enzyme
concentration.
• The initial reaction velocity is considered.
• The steady state hypothesis is followed.
Exception: Allosteric enzymes

15. Km is Michaelis Menten constant.
• It is the substrate concentration at which the reaction
velocity is half of the maximum reaction velocity.
• Significance:
• It is one unique property of enzyme and constant for a
specific substrate.
• It indicates substrate affinity.
• Low Km – higher affinity
• High Km – lower affinity

16. Turnover Number of Enzyme
• The number of substrate molecules converted to product in a unit
time by one single molecule of enzyme when it is fully saturated.
• Under standard condition, Vmax is equal to its turnover number.
• Enzymes with higher turnover numbers are more efficient.

17. Lineweaver Burk Plot
• Also known as Double Reciprocal Plot.
Advantages over MM plot:
• Determination of Km and Vmax is accurate.
• Explanation of different enzyme inhibition is possible.

18. Mechanism of Enzyme catalysis
• The enzyme catalysis and its specificity are best explained by
ACTIVE SITE or The CATALYTIC SITE.
• Extremely specific for substrate binding
• Complex three dimensional form and shape
• Nonpolar crevice
The Enzyme substrate complex formation are of two types:
1. Fischer’s Lock and Key Model
2. Koshland’s Induced Fit Model.

19. Fischer’s Lock and Key Model
• The active site is a rigid preshaped template.
• Already in proper conformation even in absence of substrate.
• Substrate fits in the active site of the enzyme just like a key fits in
a lock (structural complementarity).
• This model cannot explain the allosteric modulators

20. Koshland’s Induced Fit Model
• Here, the active site conformation is flexible and dynamic.
• Substrate binds to the enzyme to form ES complex intermediate.
• Substrate binding to the enzyme induces conformational change in the
active site to attain the final catalytic shape.
• The enzyme in turn induces a reciprocal change in substrate to alter the
configuration to transition state.

21. Enzymology (part III)
• What are the different types of Enzyme inhibition?
• What is Competitive Enzyme Inhibition? What are the kinetic
changes? Give examples. Illustrate with Double reciprocal plot.
• What is Non-competitive Enzyme Inhibition? What are the kinetic
changes? Illustrate in Double reciprocal plot.
• What is Irreversible Enzyme Inhibition? What are the kinetic changes?
Give examples. Illustrate in Double reciprocal plot.
• What is suicidal enzyme inhibition? Give examples.

22. Types of Enzyme Inhibition
Reversible Irreversible
Competitive Noncompetitive Uncompetitive

23. Reversible Competitive Inhibition
• The inhibitor is structural analogue of the substrate.
• It competes with the substrate for binding at the active site.
Kinetic changes:
By increasing the substrate concentration, inhibition can be reversed.

24. Reversible Competitive Inhibition
• Vmax unchanged
• Km increased.

25. Reversible Competitive Inhibition
• Malonic Acid is the competitive inhibitor of Succinate
Dehydrogenase.
• Sulpher drugs contain sulphanilamide (structural analogue of
PABA)- Inhibit the enzyme Dihydropteroate synthatase.
• METHOTREXATE acts as anti-cancer drugs.
• TRIMETHOPRIM acts as antibacterial drug.
• PYRIMETHAMINE acts as antimalarial drug.
• Dicumerol WARFARIN acts as anticoagulant drug.

26. Reversible Non-Competitive Inhibition
• The inhibitor has no structural resemblance to substrate.
• It binds to to the enzyme at different to active site – induces
conformational changes in enzyme
• Substrate binding occurs – but with decreased catalytic rate – product
formation decreased.
Kinetic changes:
By increasing the
substrate concentration,
inhibition cannot be
reversed.

27. Reversible Non-Competitive Inhibition
• Vmax decreased
• Km remains unchanged

28. Reversible Uncompetitive Inhibition
• The inhibitor only binds to Enzyme-Substrate complex
Kinetic changes:
By increasing the substrate
concentration, inhibition cannot be
reversed.
• Km and Vmax both decreased

29. • Inhibitors bind covalently to enzyme’s active site – active site
modified and catalytic activity destroyed permanently.
• Either functional group destruction or stable noncovalent adduct
formation
• Two types :
oGroup specific reagents
oAffinity labels
Irreversible Inhibition
1. DIFP – Di isopropyl Fluro Phosphate
2. Iodoacetate
TPCK –
Chymotrypsin Inhibitor

30. • Here the inhibitor is the modified substrate.
• Mechanism :
• The inhibitor (substrate) binds an enzyme active site.
• It is initially processed by the normal catalytic mechanism.
• The mechanism of catalysis then generates a chemically reactive intermediate
that inactivates the enzyme through covalent modification.
• Application:
• In rational drug design – to optimize drug specificity and reduce side effects
• Example :
Allopurinol
Aspirin, Ornithine Decarboxylase, MAO inhibitors
Suicide Inhibition / Mechanism based Inhibition

31. 1. What are the different mechanisms to regulate enzyme activity?
2. What is allosteric regulation of enzyme activity? Illustrate with
examples and graphical representation.
3. What are the different covalent modification which regulate
enzyme activity?
4. What is long term regulation of enzyme activity?
5. What is feedback Inhibition? Give examples.
Enzymology (part IV)

32. Regulation of Enzyme Activity
Regulation through
conformational changes
Regulation through changes in
amount of enzyme
Regulation of metabolic
pathways
By Allosteric
enzyme
By Covalent
Modification
By Protein-Protein
interaction
Proteolytic
Cleavage
Regulated Enzyme
Synthesis
Regulated Protein
Degradation
Regulation of Rate
Limiting Step
Feedback Inhibition
Tissue isoenzymes of
regulatory enzymes
Compartmentation
Counter regulatory pathways

33. Allosteric Regulation of Enzyme Activity
• Allosteric activators and inhibitors (allosteric effectors) are compounds that bind to the
allosteric site (a site separate from the catalytic site)
• It cause conformational change that affects the affinity of the enzyme for the substrate.
COOPERATIVITY IN SUBSTRATE BINDING TO ALLOSTERIC ENZYMES
• Usually an allosteric enzyme has multiple interacting subunits
• They exist in active and inactive conformations
• The allosteric effector promotes or hinders conversion from one conformation to another.
• The binding of substrate to one subunit facilitates the binding of substrate to another
subunit.
• The first substrate molecule has difficulty in binding to the enzyme because all of the
subunits are in the conformation with a low affinity for substrate (the taut “T”
conformation)
• The first substrate molecule to bind changes its own subunit and at least one adjacent
subunit to the high-affinity conformation (the relaxed “R” state.)
• The change in one subunit facilitated changes in all four subunits, and the molecule
generally changed to the new conformation in a concerted fashion.

34. Allosteric Regulation of Enzyme Activity

35. ALLOSTERIC ACTIVATORS AND INHIBITORS
• Activators bind at the allosteric site, a site physically separate from the
catalytic site. The binding changes the conformation of the catalytic
site in a way that increases the affinity of the enzyme for the substrate.
• Activators bind more tightly to the high-affinity R state of the enzyme
than the T state (i.e., the allosteric site is open only in the R enzyme)
Thus, the activators increase the amount of enzyme in the active state,
thereby facilitating substrate binding in their own and other subunits.
• In contrast, allosteric inhibitors bind more tightly to the T state.
Allosteric Regulation of Enzyme Activity

36. Allosteric Regulation of Enzyme Activity

37. Allosteric Regulation of Enzyme Activity

38. Covalent Modification
PHOSPHORYLATION • Phosphorylation by a protein kinase or
dephosphorylation by a protein phosphatase.
• Phosphate is a bulky, negatively charged
residue that interacts with other nearby amino
acid residues - conformational change in active
site.
• The conformational change makes certain
enzymes more active and other enzymes less
active.
• The effect is reversed by a specific protein
phosphatase that removes the phosphate by
hydrolysis.
• Acetylation
• ADP Ribosylation

39. Covalent Modification by Phosphorylation

40. Protein – Protein Interaction
• Calcium – Calmodulin family of Modulator protein
• Small G protein

41. Proteolytic Cleavage
• Many proteolytic enzymes are secreted as Proenzymes, which are
precursor proteins.
• They undergo proteolytic cleavage to become fully functional
(conformational changes to expose the active site).
• Chymotrypsinogen is stored in vesicles within pancreatic cells until
secreted into ducts leading to the intestinal lumen. In the digestive
tract, chymotrypsinogen is converted to chymotrypsin by the
proteolytic enzyme trypsin, which cleaves off a small peptide from
the N-terminal region (and two internal peptides).
• As the process is irreversible, specific inhibitor is required to
inactivate it.

42. Regulation through changes in
amount of enzyme
Protein synthesis begins with the process of gene transcription. The
code in messenger RNA is then translated into the primary amino acid
sequence of the protein.
• Generally the rate of enzyme synthesis is regulated by increasing or
decreasing the rate of gene transcription, processes generally
referred to as induction (increase) and repression (decrease).
The content of an enzyme in the cell can be altered through selective
regulated degradation as well as through regulated synthesis.
• All proteins in the cell can be degraded with a characteristic half-life
• Protein degradation via two specialized systems, Proteosomes and
Caspases

43. Regulation of Enzyme Activity
Regulation through
conformational changes
Regulation through changes
in amount of enzyme
Regulation of metabolic
pathways
Regulation of Rate Limiting Step
Feedback Inhibition
Tissue isoenzymes of
regulatory enzymes
Compartmentation
Counter regulatory pathways
Regulated Enzyme
Synthesis
Regulated Protein
Degradation
By Allosteric
enzyme
By Covalent
Modification
By Protein-Protein
interaction
Proteolytic
Cleavage

44. Enzymes : Its Clinical Importance
• Plasma Functional Enzymes:
• Actively secreted in the blood in appropriate amount for their
desired function in body.
• Eg – Blood coagulation enzymes.
• Plasma Nonfunctional Enzymes:
• These enzymes have no definitive function in body. They are only
released from their location during normal wear and tear.
• They are commonly used in diagnostic purposes.

45. ISOENZYMES
• Physically distinct molecular form of same enzyme with same catalytic activity
but with different physical and chemical properties.
• Oligomeric with multiple subunits - Homomultimeric & Heteromultimeric.
• Only oligomeric form is active.
• Properties :
oOrigin
oDistribution
oTemperature stability
oElectrophoretic mobility
oKm values for substrate
oInhibitor sensitivity
• Importance:
• Separation of Isoenzymes

46. Lactate Dehydrogenase
• Five isoenzymes with different subunit composition (tetramer )
• Two types of subunit – H and M – encoded by different gene
• Function – catalyzes interconversion of pyruvate and lactate using
NADH/NAD+ as coenzyme.

47. Creatine Phosphokinase
• Three Isoenzymes (Dimer)
• Two Subunits : B & M
• Function : Formation of creatine phosphate from creatine using ATP.

48. • LDH
• CPK
• Transaminases
• Alkaline Phosphatase
• Acid Phosphatase
• Amylase and Lipase
• Streptokinase
• Pepsin and Trypsin
Restriction
Endonuclease
• Glucose Oxidase
• Peroxidase
Enzymes : Its Clinical Importance
Therapeutic purposesDiagnostic purposes
Used in Laboratory as reagent
Research purposes

50. 1. Lineweaver Burk Plot is commonly known as
a) Double Logarithmic plot
b) Double Parabolic plot
c) Double Reciprocal plot
d) Double Binomial plot

51. 2. Michaelis Menten Equation is
Vo + [S]
Km [S]
c. Vmax =
Vmax [S]
Km + [S]
a. Vo =
Vmax [S]
Km + [S]
b. Vo =
Km [S]
Vo + [S]
d. Vmax =

52. 3. Induced Fit Model was proposed by
a) Fischer
b) Koshland
c) Michaelis
d) Einstein

53. 4. Dicoumarol is structural analogue of
a) Folic Acid
b) Vitamin K
c) PABA
d) Succinic Acid

54. 5. Allopurinol inhibits the enzyme Xanthine
Oxidase. This is a type of
a) Competitive inhibition
b) Noncompetitive Inhibition
c) Uncompetitive Inhibition
d) Irreversible Inhibition

55. 6. The enzyme Phosphoglucomutase falls
under the class
a) Class 4
b) Class 3
c) Class 5
d) Class 6

56. 7. International Unit of enzyme activity is
a) Amount of enzyme catalyzing the transformation of one
micromole of substrate into product in one minute.
b) Amount of enzyme catalyzing the transformation of one
mole of substrate into product in one minute.
c) Amount of enzyme catalyzing the transformation of one
micromole of substrate into product in one second.
d) Amount of enzyme catalyzing the transformation of one
mole of substrate into product in one second.

57. 8. Organophosphate compound inhibits the
Acetylcholinesterase by
a) Competitive inhibition
b) Noncompetitive Inhibition
c) Uncompetitive Inhibition
d) Irreversible Inhibition

58. 9. What type of enzyme inhibition is
represented in the double reciprocal plot ?
a) Competitive inhibition
b) Noncompetitive
Inhibition
c) Uncompetitive
Inhibition
d) Irreversible Inhibition

59. 10. One of the assumption based on which
Michaelis Menten Equation is formulated is
a) Km = [S] when V0 = Vmax
b) Rates of formation and breakdown of Enzyme-substrate
complex are equal
c) In competitive enzyme inhibition, Vmax remaiins
unchanged.
Vmax [S]
Km + [S]
d) Vo =

60. 11. Catalase is an example of
a) Coenzyme
b) Proenzyme (Zymogen)
c) Metallozyme
d) Non-functional enzyme

61. 12. The relation between Katal and SI unit of
enzyme activity is
a) 1 Katal = 6 x 107 IU
b) 1 Katal = 7 x 106 IU
c) 1 IU = 6 x 107 Katal
d) 1 IU = 7 x 106 Katal

62. 13. Turnover Number of Enzyme is
a) The number of substrate molecules converted to product in a unit
time by one mole of enzyme when it is fully saturated.
b) The number of enzyme molecules required to convert 1 mole of
substrate to product in a unit time when enzyme is fully
saturated.
c) The number of substrate molecules converted to product in a unit
time by one single molecule of enzyme when it is fully saturated.
d) The number of enzyme molecules required to convert 1
micromole of substrate to product in a unit time when enzyme is
fully saturated.

63. 14. Enzyme
a) Increases activation energey
b) Decreases activation energy
c) Increases free energy
d) Decreases free energy

64. 15. If temperature is increased beyond
optimal temperature, the enzyme activity is
decreased because
a) The substrate is dissociated from the enzyme
b) The enzyme is denatured
c) The product causes feedback inhibition
d) The substrate is melted.

65. 16. All are enzyme kinetic plots except
a) Eadie Hofstee Plot
b) Michaelis Menten Plot
c) Lineweaver Burk Plot
d) Ramachandran Plot

66. 17. In competitive inhibition
a) Vmax unchanged, Km decreases
b) Vmax decreases, Km decreases
c) Vmax decreases, Km increases
d) Vmax increases, Km unchanged

67. 18. Allosteric activators
a) Induces the stability of ‘T’ form of enzyme
b) Binds to the active site of the enzyme
c) Re-orient the alignment of amino acids surrounding
the active site
d) Denatures the enzyme

68. 19. Advantage of Lineweaver Burk plot over
MM plot is
a) Allosteric regulation of enzymes can be studied
b) Estimation of Km and Vmax is more accurate
c) Initial and terminal velocity – both can be
considered
d) Pre-steady state kinetics can be studied more
precisely

69. 20. Activation and deactivation of enzyme by
Protein Kinase and Phosphatase is a type of
modification of
a) Electrostatic bond
b) Covalent bond
c) Hydrogen bond
d) James Bond