Chemistry 501 Handout 6 Enzymes Chapter 6 Dep. of Chemistry & Biochemistry Prof. Indig Chemistry 501 Handout 6 Enzymes Chapter 6 Lehninger. Principles of Biochemistry. by Nelson and Cox, 5th Edition; W.H. Freeman and Company

Complete, catalytically active enzyme: holoenzyme With exception of a small group of catalytic RNA molecules, all enzymes are proteins Largest class of proteins. More than 3000 enzymes known. Enzymes are biological catalysts that accelerate reactions. Enzymes are generally highly specific and react with only one substrate to form one product and can enhance reaction rates by as much as 1017. Some enzymes require cofactors / coenzymes for activity Coenzymes act as transient carriers of specific functional groups A cofactor or coenzymes that is very tightly or even covalently bound to the enzyme protein is called a prosthetic group. Complete, catalytically active enzyme: holoenzyme Only protein part (without cofactor and/or coenzyme): apoenzyme or apoprotein

Enzymes are classified by the reactions they catalyze International Classification System (nomenclature): (Four-part classification number and a systematic name) Systematic name: ATP:glucose phosphotransferase Classification number: 2.7.1.1. 2. --> Transferase (class) 7. --> phosphotransferase (subclass) 1. --> phosphotransferase with a hydroxyl group as acceptor 1. --> D-glucose as the phosphoryl group acceptor ATP + D-glucose --> ADP + D-glucose-6-phospate Hexokinase

Binding of a substrate to an enzyme at the active site Representation of a simple enzymatic reaction Enzymes affect reaction rates, not equilibria E + S  ES  EP  E + P Reaction coordinate diagram for a chemical reaction Reaction coordinate diagram comparing enzyme-catalyzed and uncatalyzed reactions

S  P S → P Equilibrium Reaction Reaction rate = V = k [S] If the rate depends only on the concentration of S (first-order) From the Transition-State Theory:

Weak interactions between enzyme and substrate are optimized in the transition state The energy derived from enzyme-substrate interaction is called binding energy, DGB Enzyme active sites are complementary not to the substrate per se, but to the transition state through which substrates pass as they are converted into products during an enzymatic reaction Complementary shapes of a substrate and its binding site on the enzyme Role of binding energy in catalysis An imaginary enzyme (sticase) designed to catalyze breakage of a metal stick

How a catalyst circumvents unfavorable charge development during cleavage of a amide Rate enhancements by entropy reduction Specific acid-base catalysis General acid-base catalysis Hydrolysis of an amide bond - same reaction as that catalyzed by chymotrypsin and other proteases

formation of covalent bonds with substrates Amino acids in general acid-base catalysis Covalent and general acid-base catalysis First step of the reaction His57 His57 Amino acid side chains and the functional groups of some cofactors can serve as nucleophiles in the formation of covalent bonds with substrates

Enzyme Kinetics Michaelis-Menten kinetics initial rate measurements Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction Michaelis-Menten kinetics initial rate measurements [S] >> [E] [ES] ~ constant V0 = K2 [ES] Michaelis constant

When km > > [S] When [S] > > km Dependence of initial velocity on substrate concentration When km > > [S] When [S] > > km

A double-reciprocal or Lineweaver-Burk plot Lineweaver-Burk equation

Kcat describes the limiting rate of any enzyme-catalyzed reaction at saturation

Many enzymes catalyze reactions with two or more substrates Common mechanisms for enzyme-catalyzed bisubstrate reactions

Pre-steady state kinetics can provide evidence for specific reaction steps Ternary complex is formed in the reaction (as indicated by the intersecting lines) Ping-Pong (double displacement) reaction

Enzymes are subject to reversible and irreversible inhibition

Enzymes are subject to reversible and irreversible inhibition

Irreversible inhibition Reaction of chymotrypsin with diisopropylfluorophosphate irreversibly inhibits the enzyme Suicide inactivators or mechanism-based inactivators

Three polypeptide chains linked Examples of Enzymatic Reactions Chymotrypsin is a protease specific for peptide bonds adjacent to aromatic amino acids (Trp, Tyr, Phe) Key active-site amino acid residues Ser195, His57, and Asp102 Aromatic amino acid side chain Pocket in which the amino acid side chain of the substrate is bound Three polypeptide chains linked by disulfide bonds

The chymotrypsin mechanism involves acylation and deacylation of a Ser residue

Regulatory Enzymes Allosteric enzymes undergo conformational changes in response to modulator binding Conformational change Subunit interactions in an allosteric enzyme, and interactions with inhibitors and activators

Two views of the regulatory enzyme aspartate transcarbamoylase This allosteric regulatory enzyme has two stacked catalytic clusters, each with three catalytic polypeptide chains (in shades of blue and purple) and three regulatory clusters, each with two regulatory peptide chains (in red and yellow). Modulator binding produces large changes in enzyme conformation and activity

In many pathways a regulatory step is catalyzed by an Feedback inhibition Buildup of the end product ultimately slows the entire pathway In many pathways a regulatory step is catalyzed by an allosteric enzyme Example of heterotropic allosteric inhibition

Phosphoryl groups affect the structure and catalytic activity of enzymes Regulation of muscle glycogen phosphorylase activity by multiple mechanisms

Activation of zymogens by Some enzymes and other proteins are regulated by proteolytic cleavage of a precursor Activation of zymogens by proteolytic cleavage