ENZYMES

Dindin H. Mursyidin
Laboratory of Genetics & Molecular Biology
dindinhm@gmail.com
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
Multiple subunits --
more control
MORE SUBUNITS -- MORE CONTROL II

Inhibition
MORE SUBUNITS -- MORE CONTROL III

Allosteric enzyme (hemoglobin)

Monomeric enzyme
(myoglobin)

Cooperative binding of O2
MORE REGULATION OF PROTEIN FUNCTION

 Reversible covalent modification.

Phosphorelays.

 Interaction with control proteins.

Calmodulin.

 Compartmentalization.
 Proteolytic activation.
Common in control of proteases.
CONTROL BY PROTEOLYTIC CLEAVAGE

chymotrypsinogen chymotrypsin
HIV PROTEASE AND PROTEOLYTIC
CONTROL

HIV protease is similar to mammalian aspartic

proteases.
Two aspartates in the active site.
Pre-protein must be cleaved for activity.

But, HIV protease is a homodimer (symmetrical).

Symmetric transition state analogs inhibit HIV

protease much more than mammalian enzymes.

HIV protease inhibitors represent a great success of

structure-based drug design.