Enzymes: Dr. Dindin H. Mursyidin, M.SC

ENZYMES
Dr. Dindin H. Mursyidin, M.Sc.

Laboratory of Genetics & Molecular Biology
Faculty of Mathematics and Natural Sciences
University of Lambung Mangkurat
MAIN TOPICS
 Properties of enzymes
 Thermodynamics and kinetics
 Regulation of enzyme activity

SOME COMMON TYPES OF ENZYMES
Hydrolases Polymerases
Nucleases Kinases
Proteases Phosphatases
Synthases ATP-ases
Isomerases Oxidoreductases (dehydrogenases)
OXIDATION-REDUCTION REACTIONS
(REDOX)
 Oxidation = loss of electrons
 Reduction = gain of electrons
 Must be balanced
 Very common in biology

PROPERTIES OF ENZYMES
 Enzymes are highly efficient and specific catalysts.
 Enzymes alter rates, not equilibria.
 Enzymes stabilize transition states.
 Reaction rates depend on concentrations of enzymes,

substrates, and on the efficiency of the enzyme.
ENZYME ACTIVE SITES
Active sites:
The region that binds substrate.
Only a small fraction of the enzyme.
Formed from AAs in different parts of the sequence.
carboxypeptidase
ENZYME ACTIVE SITES
Active sites:
Usually form a cleft or pocket.
Substrates are bound by

multiple weak interactions.
REACTION THERMODYNAMICS
Consider:
[A][B] [C][D]
[C][D]
Keq =
[A][B]
Keq depends only on the nature of the products and the

reactants.
Rxn. will proceed spontaneously only when the change in
free energy (G) is negative.
REACTION THERMODYNAMICS II
Enzymes stabilize the transition state, lowering the activation barrier.

ACID-BASE CATALYSIS
ENZYME KINETICS
KINETICS: VMAX AND KM
Reaction rate (V) varies with substrate concentration.
Vmax = the maximum reaction rate.
Km = substrate concentration where V = Vmax/2
Measures affinity of enzyme for substrate.
[S]
[S] + Km The Michaelis-Menten equation
V = Vmax
VMAX AND KM
Vmax depends on the amt. of enzyme.

DIFFERENT SUBSTRATES, VMAX AND KM
Km is a property of both enzyme and substrate.

TO LINEARIZE MICHAELIS-MENTEN
Lineweaver-Burk plots
1/V V and [S] can be

slope = Km/Vmax
determined
experimentally.
1/Vmax
-1/Km
1/[S]
Can also do Eadie-Hofstee (V vs. V/[S]) or Haynes ([S]/V vs. [S])

plots.
ENZYMATIC EQUATIONS
E+S ES EP E+P
There are at least three steps…….

ENZYMATIC EQUATIONS II
E+S ES EP E+P
k1 kcat
E+S ES E+P
k2
Km = (k2+ kcat )/k1
usually kcat <<< k2, so Km = k2/k1 = Kd

Catalytic efficiency and proficiency
Catalytic efficiency = kcat / Km
kcat / Km
Catalytic proficiency =
knon
kcat = # substrate molecules converted per second
Km = affinity of enzyme for substrate
Catalytic (kinetic) perfection: Efficiency = diffusion rate (~108 M-1S-1)

Catalytic efficiency and proficiency II
Protein / inositol phosphatases

Enzyme Phosphorylated substrate kcat (s−1) kcat/Km (M−1⋅s−1)
PP1 Phosphoryl-phosphorylase a 39 4 × 106

FBPase Fructose 1,6-bisphosphate 21 1.5 × 107
IP Inositol-1-phosphate 22 3 × 105
None MeP and compound 1 2 × 10−20 (knon)
Lad, Chetan et al. (2003) Proc. Natl. Acad. Sci. USA 100, 5607-5610
Copyright ©2003 by the National Academy of Sciences

Catalytic efficiency and proficiency II
Protein / inositol phosphatases

Enzyme Phosphorylated substrate kcat (s−1) kcat/Km (M−1⋅s−1)
PP1 Phosphoryl-phosphorylase a 39 4 × 106

FBPase Fructose 1,6-bisphosphate 21 1.5 × 107
IP Inositol-1-phosphate 22 3 × 105
None MeP and compound 1 2 × 10−20 (knon)
Lad, Chetan et al. (2003) Proc. Natl. Acad. Sci. USA 100, 5607-5610
Copyright ©2003 by the National Academy of Sciences

Catalytic
efficiency and
proficiency III
ENZYME INHIBITION
Tool to study enzymatic reactions.

Important in host/pathogen interactions.
Important in drug design.
Irreversible (suicide) inhibition (eg - nerve gas).

Reversible inhibition:
competitive (eg. - transition state analogues).
non-competitive.
uncompetitive.
Type of inhibition can be determined experimentally.
ENZYME INHIBITION IN THE LAB
Competitive
Inhibitor binding prevents substrate binding.

1/V + inhibitor
May be active site binding or not.
no inhibitor
Very common.
1/Vmax
1/[S]
Vmax doesn’t change.

Non-competitive
1/V + inhibitor
Inhibitor binding alters the enzyme.
-1/Km no inhibitor Substrate can still bind.
Binding must be away from active site.

1/[S]
Also common.
Km doesn’t change.
Un-competitive
Inhibitor binds ES complex.
Works best when S is high.
Rare.
Km and Vmax both change

REGULATION OF PROTEIN
FUNCTION BY ALLOSTERIC
CONTROL
= noncovalent interaction away from the active site.

Protein-protein interactions.
Small molecules.
Common in multi-subunit protein complexes.
Allosteric enzymes do not obey Michaelis-Menten kinetics.

Feedback inhibition
-- allosteric control
Feedback is usu. at the first

committed step in the pathway.
POSITIVE FEEDBACK REGULATION
NEGATIVE FEEDBACK INHIBITION

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ENZYMES

Dr. Dindin H. Mursyidin, M.Sc.

 Thermodynamics and kinetics

 Regulation of enzyme activity

 Very common in biology

 Enzymes are highly efficient and specific catalysts.

 Enzymes alter rates, not equilibria.

 Enzymes stabilize transition states.

 Reaction rates depend on concentrations of enzymes,

Substrates are bound by

Keq depends only on the nature of the products and the

Enzymes stabilize the transition state, lowering the activation barrier.

Reaction rate (V) varies with substrate concentration.

Vmax = the maximum reaction rate.

Km = substrate concentration where V = Vmax/2

Measures affinity of enzyme for substrate.

Vmax depends on the amt. of enzyme.

Km is a property of both enzyme and substrate.

1/V V and [S] can be

Can also do Eadie-Hofstee (V vs. V/[S]) or Haynes ([S]/V vs. [S])

There are at least three steps…….

Km = (k2+ kcat )/k1

usually kcat <<< k2, so Km = k2/k1 = Kd

Catalytic efficiency = kcat / Km

Km = affinity of enzyme for substrate

Catalytic (kinetic) perfection: Efficiency = diffusion rate (~108 M-1S-1)

Protein / inositol phosphatases

PP1 Phosphoryl-phosphorylase a 39 4 × 106

Copyright ©2003 by the National Academy of Sciences

Protein / inositol phosphatases

PP1 Phosphoryl-phosphorylase a 39 4 × 106

Copyright ©2003 by the National Academy of Sciences

Tool to study enzymatic reactions.

Irreversible (suicide) inhibition (eg - nerve gas).

Inhibitor binding prevents substrate binding.

Vmax doesn’t change.

-1/Km no inhibitor Substrate can still bind.

Binding must be away from active site.

Inhibitor binds ES complex.

Works best when S is high.

Km and Vmax both change

= noncovalent interaction away from the active site.

Common in multi-subunit protein complexes.

Allosteric enzymes do not obey Michaelis-Menten kinetics.

Feedback is usu. at the first

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