4 enzyme mechanism 20140906

Mechanisms of enzymes

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

(Transition state)

Activation energy

(required for reactants to achieve transition state)

Effect of an enzyme on a chemical reaction

- Lowering of activation energy - More reactants (= substrates) achieve transition state - Acceleration of reaction

•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.

How do enzymes reduce the activation energy?

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

- 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

• 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:-)

Base catalysis:

• Removes a proton from water

• Generates an OH- equivalent which attacks the carbonyl carbon

• Cleavage of C-N bond

1.

2.

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 His-159 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

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

Acid-base catalysis and covalent catalysis for chymotrypsin

Chymotrypsin

Peptide substrate

scissile bond

specificity pocket

Structural similar serine proteases with different substrate specificities

Chymotrypsin Trypsin Elastase

Specificity pockets of three serine proteases:

C

O

Cα

(attacked by Ser-195)

N