Enzymes

Learning Objectives
General properties of enzymes How enzymes accelerate chemical reactions Mechanisms of enzymatic catalysis Examples: lysozyme and serine proteases Inhibition and regulation of enzymes

What are Enzymes?

From ancient times, people use yeast to produce alcohol from sugar (a fermentation process). Chemists of XIX century failed to reproduce reactions of the fermentation process by combining pure reagents. The term enzymes (Greek: enzumos, in yeast) was proposed to hint that something inside the yeast catalyzes reactions of fermentation. Catalysts are substances that increase the rate of a chemical reaction without being consumed in the chemical process. Today we know that most enzymes are proteins. However, certain RNAs also demonstrate catalytic properties.

General Properties of Enzymes

Most biomolecules have functional groups that, in principle, may react with each other. Cells avoid a chemical mess because the rates of uncatalyzed reactions are very low. The rates of enzymatically catalyzed reactions are 106 to 1012 times greater than those of the corresponding uncatalyzed reactions. Enzymatically catalyzed reactions occur at mild conditions.

General Properties of Enzymes

Enzymes have a great degree of specificity relative to their substrates and products. Therefore, enzymatic reactions rarely have side products. Enzymatically catalyzed reactions are regulated by allosteric mechanisms, covalent modification of enzymes, and variation of the amount of the synthesized enzymes.

Nomenclature
Enzymes are named by appending the suffix -ase to the name of the corresponding substrate or chemical reaction. For example, urease catalyzes the hydrolysis of urea; alcohol dehydrogenase catalyzes oxidation of alcohols to aldehydes. Classification of enzymes according to reaction type: Classification
Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases

Type of reaction catalyzed

Oxidation-reduction Transfer of functional groups Hydrolysis Group elimination to form double bonds Isomerization Bond formation coupled with ATP hydrolysis

Substrate Specificity
Fig. 11-1 A binding site for a substrate is usually located in a cleft (pocket, gorge, crevice) on the protein surface. The shape of a substrate has a geometric complementary to the shape of the binding site. Amino acid residues in the active site of an enzyme are arranged to maximize favorable electrostatic interactions and Hbonds with the corresponding substrate (electronic complementarity).

Substrate Specificity
Enzymes may undergo certain conformational changes upon substrate binding (induced fit). Enzymes are highly specific to the configuration of their substrates. Certain chemical reactions are catalyzed by corresponding enzymes only in the presence of cofactors (metal ions) or coenzymes (organic molecules).

Free Energy of Activation

G Transition state X# G# G#

Chemical reaction: A + B GR, free energy of reactants

X#

C+D

GP, free energy of products G#, free energy of transition state X# G# = G# - GR is free energy of activation Reaction rate is proportional to
G reaction = GP - GR < 0

G # / RT

GR GP Reactants A+B Products C+D

Reaction coordinate

Enzymes Reduce Free Energy of Activation

G

Uncatalyzed reaction Catalyzed reaction

G#, the reduction of G# by the enzyme The rate enhancement =
A+B C+D A+B C+D
G # / RT

G# is the same for the forward and reverse reactions. Reaction coordinate

A 10-fold rate enhancement requires G# 5.7 kJ/mol

Curved Arrow Convention

Curved arrows indicate electron pairs rearrangements during chemical reaction. Arrows start at the reactants electron pairs and point to the electron-deficient centers that attract the electron pairs Example: R .. R-NH2 + C=O R Amine Aldehyde or ketone R .. R-N-C-OH H R Carbinolamine intermediate H+
+

R R-N=C H R + H2O

Imine (Schiff base)

Catalytic Mechanisms
Uncatalyzed keto-enol tautomerization General acid catalysis: partial transfer of H+ from an acid General base catalysis: partial withdrawing of H+ by a base

Fig. 11-8

Catalytic Mechanisms
Metal ion catalysis:
- Proper orientation of the substrate by a metal ion - Reversible changes in oxidation states of a metal ion - Electrostatic stabilization (e.g., shielding of negative charges) About one-third of all known enzymes require the presence of metal ions for catalytic activity

Fig. 11-13

Catalytic Mechanisms
Covalent catalysis - transient covalent bond with the substrate Electrostatic catalysis - lowering G# by enzymes charges. Proximity and orientation effects: - bringing substrates into contact with their catalytic groups - properly orienting of substrates - freezing translational and rotational motions of substrates Preferential binding of the transition state complex - an enzyme has maximal affinity to the transition-state structure of the corresponding substrate

Polysaccharide Component of Bacterial Cell Walls

Fig. 11-16

Lysozyme
- an enzyme with an antiseptic activity Fig. 11-17 - occurs in human tears, saliva, and other body fluids - catalyzes hydrolysis of (14) glycosidic bonds in the cell walls of certain bacteria.

Lysozyme
The active site comprises protonated Glu35 and deprotonated Asp52. Substrate binds in a strained conformation (catalysis by preferential binding of the transition state) to the enzymes active site.

Fig. 11-18

Lysozyme
Glu35 transfers its proton to the glycosidic-bond oxygen and cleaves the bond (general acid catalysis). E ringproduct leaves the enzyme.

Fig. 11-21

Lysozyme

Asp52 binds to the carbocation (covalent catalysis)

Lysozyme
Water molecule hydrolyzes the covalent bond between Asp52 and substrate. Glu35 helps water to dissociate (general base catalysis)

Lysozyme
The D ring-product leaves the enzyme. The catalytic cycle is completed

Serine Proteases
Fig. 11-26

- proteolytic enzymes having a common catalytic mechanism that involves a Ser residue - include digestive enzymes and other enzymes involved in numerous biochemical processes.

Serine Proteases
Chymotrypsin, trypsin, and elastase are best studied serine proteases. Each enzyme catalyzes the hydrolysis of peptide bonds depending on the nature of the residue adjacent to the scissile bond. Fig. 11-27

Chymotrypsin is specific for an aromatic hydrophobic residue (Phe, Trp, Tyr)

Trypsin is specific for a positively charged residue (Lys, Arg)

Elastase is specific for a small neutral residue (Gly, Ala, Ser, Val).

Inhibition of Enzymes
Enzyme inhibitors are substances that bind (reversibly or irreversibly) to an enzyme and reduce the enzymes activity. Competitive inhibitors compete with substrates for the binding site at the enzyme. Irreversible inhibitors form covalent bonds with enzymes and permanently inactivate them. Many drugs and poisons are enzyme inhibitors. For example, AIDS is treated with drugs that inhibit activities of certain viral enzymes.

Regulation of Enzyme Activity

1. Control of enzyme availability The cell controls - the rate of syntheses of enzymes - the rate of degradation of enzymes 2. Control of enzyme activity Some substances change conformation of the enzyme active site via non-covalent binding at a protein site remote from the active site. The binding changes dramatically the enzymatic activity. Such substances are called allosteric effectors. Activity of some enzymes is regulated by covalent modification, such as phosphorylation and dephosphorylation of specific Ser, Thr, or Tyr residues.