Mechanisms of enzymes

Energy diagram for a single-step reaction (A-B + C A + B-C)

(Transition state)
Activation (required for reactants to
energy achieve transition state)

Effect of an enzyme on a chemical reaction

Lowering of activation energy

More reactants (= substrates) achieve transition state
Acceleration of reaction

How do enzymes reduce the activation energy?

Enzyme binding of substrates increases the initial ground state of

the enzyme-substrate complex (brings reactants in close proximity
and into correct orientation, thus more transition state formation)

Stabilize the transition state (by tight binding) to lower activation

energy barrier.

Induced Fit
-

Substrate-induced conformation change

A substrate specificity effect
Example: Hexokinase

Open form: no substrates

Closed form: with bound glucose

- Exclusion of water
- No hydrolysis of ATP

When an enzyme binds to the appropriate substrate, subtle

changes in the active site occur. This alteration of the active site
is known as an induced fit. Induced fit enhances catalysis, as the
enzyme converts substrate to product.
Release of the products restores the enzyme to its original form.
The enzyme can repeat this reaction over and over, as long as
substrate molecules are present.

- Not a simple lock and key model

- Dynamic interaction between enzyme and
substrates

Chemical modes of enzymatic catalysis

Polar amino acid residues in active sites
- In the enzyme-substrate complex, substrates are in proximity to reactive
amino acid resides in enzyme active sites
- Enzymes usually have 2-6 catalytic residues in active site
- Ionizable side chains (in polar AA) can act as acids, bases, or nucleophiles
- Involved in substrate binding and/or transition state formation

1. Acid-Base Catalysis
- A proton is transferred between the enzyme and the substrate
- Amino acid side chains that can act as acid-base catalysts:

- Serving as proton donor or accepters depending on their protonation status

Base catalysis:
1.

Involves AA residues that can accept a proton

Can remove proton from OH, -NH, -CH, etc. and cause bond
cleavage
Creates a strong nucleophillic reactant (i.e. X:-)
2.

Removes a proton from water

Generates an OH- equivalent which attacks the carbonyl carbon
Cleavage of C-N bond

Acid catalysis
-

Proton donation
A covalent bond may break more easily if one of its atom is protonated

BH+

2. Covalent Catalysis (nucleophilic catalysis)

20% of all enzymes employ covalent catalysis

A-X + B + E

BX + E + A

A group from a substrate binds covalently to enzyme

The intermediate enzyme substrate complex (A-X) then donates the group
(X) to a second substrate (B)

Side chains that can act as covalent catalysts:

They serve as nucleophiles in the deprotonated forms

Effect of pH on enzyme reaction rates

- Ionizable side chains of catalytic AA residues require proper pH for
achieving the necessary protonated status for catalysis

- Catalytic cysteine-25 and histidine-159 residues in papain (a papaya

protease):

The activity of papain depends on His159 and Cys-25 in the active site

General acid-base catalysis mechanism for

triose phosphate isomerase

Formation of enzyme-bound intermediate:

Glu-165: a general acid-base catalyst

His-95: shuttles a protein between the
oxygen atoms of an enzyme-bound
intermediate

Acid-base catalysis and covalent catalysis for chymotrypsin

Chymotrypsin
- cleavage of peptide bond
- a serine protease
- Catalytic triad: His-57, Asp-102, Ser-195

scissile
bond

specificity
pocket
Peptide substrate

Structural similar serine proteases with different substrate specificities

Chymotrypsin

Trypsin

Specificity pockets of three serine proteases:

(attacked by
Ser-195)

O
C
C
N

Elastase