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.

 ENZYME INHIBITION IN THE LAB

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.
ENZYME INHIBITION IN THE LAB

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