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Dr. Sanjeeva Srivastava

IIT Bombay

Enzymes basic concepts

Enzyme kinetics, energetic, inhibition

Catalytic strategies
Regulatory strategies

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Proteomics Course

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Enzymes are molecular catalysts

Almost all known enzymes are proteins
Enzymes accelerate reaction million folds
Enzymes are highly specific, catalyzes
single or closely related reactions

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Trypsin
Specific, cleaves bond only on carboxyl side of Lys/Arg

Thrombin
Hydrolysis of Arg/Gly bonds in specific peptide
sequences

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Proteomics Course

Enzyme class

Type of reaction

Oxidoreductase

Oxidation-reduction

Transferase

Group transfer

Hydrolase

Hydrolysis reaction

Lyase

Addition of groups to double bonds/

removal of groups to form double bonds

Isomerase

Group transfer within the molecule

Ligase

Bond formation at the expense of ATP

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NPTEL

NPTEL

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Coenzyme
Cofactor + Apoenzyme = Holoenzyme
Apoenzyme

Holoenzyme

Metal cofactor
Fe2+

Apoenzyme
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Metals Coenzyme
Loosely bound

Fe2+

Tightly bound
prosthetic group

Holoenzyme
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G must be negative for a spontaneous reaction

Energy required to convert substrates to product
(Rate of reaction)
Enzymes cannot alter reaction equilibrium
Accelerate attainment of equilibrium concentration

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Transition state [ES]

G uncatalyzed reaction

Free energy

G catalyzed reaction
Substrate [S]

Product [P]
Reaction progress

Activation energy (G) - difference in free energy between

transition state and substrate
Enzyme decreases activation energy
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Enzyme-substrate complex (ES) formation is

first step in catalysis
Uncatalyzed reaction

Substrate [S]

Transition
state

Product [P]

Enzyme catalyzed reaction

Substrate [S]

Enzyme
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ES complex
Proteomics Course

Product [P]
NPTEL

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Region of enzyme that binds to substrate

Occupies only small portion of enzyme
Extra amino acids provide scaffolding

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Substrate
Active site

Enzyme-substrate [ES]
complex

Enzyme

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Substrate

Enzyme

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Active
site

Active site
modified upon
substrate
approach

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ES complex

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Role of enzyme catalysis (V0): number of moles

of product formed per second
Michaelis-Menten explained kinetic
characteristics
An enzyme (E) that catalyzes substrate (S) to
product (P):
k1
[E] + [S]

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k-1

k2
[ES]

k-2

[E] + [P]

Proteomics Course

NPTEL

Michaelis-Menten equation
V0 = Vmax [S]
[S] + KM

Vmax reaction rate when

enzyme is fully saturated with S
KM Michaelis constant
S substrate concentration

KM is a measure of strength of ES complex

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Vmax

Vmax/2
Km

Substrate concentration [S]

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1 / Vo

Slope = Km / Vmax

1 / Vmax

- 1 / Km

1 / [S]

1
Vo
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Km
Vmax[S]
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1
Vmax
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Enzyme inhibition provides insights into catalysis

Major control mechanism
Two type of enzyme inhibition

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Reversible

Competitive

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Non-competitive

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Substrate
[S]
Enzyme
[E]

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Uncompetitive

NPTEL

Product
[P]

ES
complex

Inhibitor
[I]

No
reaction

Proteomics Course

NPTEL

12

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Enzyme
[E]

Substrate Enzyme-substrate
[S]
[ES]

Product
[P]

Inhibitor
[I]

ESI
complex

No
reaction
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Proteomics Course

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Irreversible

1. Group specific

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2. Reactive
substrates/
Affinity labeling

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3. Suicide inhibitors

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Active site contains reactive group which covalently

attaches to substrate
Example chymotrypsin

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Proteomics Course

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A molecule plays a role of a proton donor or

acceptor
Example carbonic anhydrase, histidine facilitates
removal of H+

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Metal ions serve as bridge between E and S

Increase binding energy & holding substrate in
appropriate conformation for catalysis
Example Nucleoside Monophosphate (NMP)
kinases

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Proteomics Course

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Allosteric proteins possess regulatory sites and

multiple functional sites
Allosteric proteins have property of cooperativity
Allosteric proteins are information transducers

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Isozymes catalyze the same reaction but differ in

structural characteristics
Isozymes are expressed in distinct organelle or at
distinct stages of development

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An effective method to control enzyme activity

Example: phosphorylation

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Inactive precursor is called zymogen

Mechanism involves cycle of active and inactive
states
Example - chymotrypsin, trypsin

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Proteomics Course

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Enzymes basic concepts

Catalytic strategies
Regulatory strategies

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Proteomics Course

NPTEL

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Berg J., Tmyoczko J. & Stryer L., Biochemitry fifth ed., W. H.

Freeman & company, 2002. ISBN: 0716746840.
Nelson D. & Cox M.,Lehninger, Principles of Biochemistry fourth
ed., 2004. W. H. Freeman and company. ISBN: 023022699X.
Price N., & Stevens L., Fundamentals of enzymology, third ed.,
Oxford university press, 1999, ISBN: 019850229X
Voet D. & Voet J., Biochemistry fourth ed., Wiley, 2000. ISBN:
047158651X.
Advances in Enzymology and Related Areas of Molecular Biology,
Volume 43. Binding Energy, Specificity, and Enzymic Catalysis: The
Circe Effect. Alton Meister. DOI: 10.1002/9780470122884.ch4

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