Lecture 12:Enzyme Catalysis

Catalytic Strategies

Steps in a Reaction

Enzymes are Classified According to the Reactions They Catalyse

But while the details of the particular reaction vary fromenzyme to enzyme, similar strategies are used to carry them out.

Common Types of Catalysis

Covalent Catalysis: A group on the enzyme becomes covalently modified duringreaction, e.g. by forming a covalent bond to the substrateduring the reaction.

General Acid-Base Catalysis:A group on the enzyme acts as an acid or base: it removes aproton from or donates a proton to the substrate during

the reaction.

Metal Ion Catalysis:A metal ion is used by the enzyme to facilitate a chemicalrearrangement or binding step.

Catalysis by Approximation:The enzyme holds two substrates near in space and in preciselythe correct spatial orientation to optimize their reaction.

(Most enzymes use a combination of several of these strategies)

Enzyme Substrate CovalentIntermediate

Enzyme Products

Covalent Catalysis

Rate acceleration by transient formation of a COVALENT enzyme-substrate bond

Reaction can’t go back because displaced group has been released.The enzyme alters pathway to get to product by stabilizing the intermediate with a covalent bond.

But: if a covalent bond is formed at an intermediate step, the bond must be broken in a subsequent step to finish the reaction.

Nucleophile: an electron-rich group that attacks nuclei.Examples of nucleophiles among protein functional groups:

unprotonated His imidazoleunprotonated -amino groupunprotonated sidechain amino group of Lysthiolate anion (-S-) of Cysaliphatic -OH of Sersidechain carboxylates of Glu, Asp

(Also some coenzymes.)

Covalent Catalysis Requires a Highly Reactive Group

Some chemical group that can invade the substrate- usually a nucleophile.

General Acid-Base Catalysis

Specific functional groups in enzyme structure are positioned to eitherDonate a proton (act as a general acid)or Accept a proton (act as a general base).

This enables enzyme to avoid unstable charged intermediates in reaction,so as to keep the transition state in a stable (low-energy) state

But: A group that donates a proton (acts as a general acid) in catalysis has tothen accept a proton (act as a general base) later in catalytic mechanismfor catalyst to be regenerated in original form.

Examples of general acid/base catalysts among protein functional groups:His imidazole-amino groupthiol of CysR group carboxyls of Glu, AspSidechain amino group of LysAromatic OH of TyrGuanidino group of Arg

Metal Ion Catalysis

Metal ions can be used in a variety of ways by enzymes. (In fact they are souseful that about one-third of enzymes use them for one thing or another.)

Binding and orientation of substrate:By forming strong ionic interactions with substrate, it can be

precisely oriented. (Especially strong because when water is excluded from active site, the dielectric constant is quite low.)

Redox reactions:Ions that have more than one possible chargestate (eg. iron Fe2+ and Fe3+) can gain or lose electrons duringthe reaction, avoiding unstable charged intermediates.

Shielding or stabilizing negative charges: If charge on substrate or on transition state is an integralpart of the reaction, the enzyme can form strong ionic interactionswith that charge which are stabilizing.

Enzyme Substrate

Unstable Stable

+2-

-

+2 ++3 0

Binding and orientation of substrateAND/ORShielding or stabilizing negative charges

RedoxReactions

Catalysis by Approximation

Proximity: Reaction between bound molecules doesn't require an improbablecollision of 2 molecules. They're already in "contact" (increases localconcentration of reactants).

Orientation: Reactants are not only near each other on enzyme, they'reoriented in optimal position to react. The improbability of collidingin correct orientation is taken care of.

Substrates held close in spaceand in correct orientation

Results in moreefficient reaction pathway

Other Effects that Stabilize the Transition State

Electrostatic Effects:Increase in strength of ionic interactions due to lowerdielectric constant.

Desolvation:Exclusion of water from active site.

Induced Fit:Change in conformation of enzyme or substrate tooptimize interactions.

Providing a lower dielectric constant of the environment in theactive site (hydrophobic environment)

Altering pK values of specific functional groups.

Stabilizing a particular conformation of the critical groups inthe active site by electrostatic interactions.

Stabilizing (binding) a charged intermediate or transition state byproviding an oppositely charged enzyme group close by.

Electrostatic Effects

Enhancement of the attraction between opposite chargesby various means.

Examples:

221

Dr

qqkF Coulomb’s Law:

Lower dielectric constant environment than in water.results in stronger electrostatic interactions.

Reactive groups of reactants are protected from H2O,so H2O doesn't compete with reactants or affect equilibrium.

Desolvation

By sequestering reactants away from water, two major effects areAchieved:

Conformational change can:

Promote faster chemical steps(eg by orienting catalytic groups on enzyme

Promote tighter transition state binding(eg by orienting binding groups on enzyme)

Close off active site and sequester reactants away from water.

Induced Fit

Conformational change resulting from substrate binding may stabilize a different conformation of either enzyme or substrate or both.

Examples:

Chymotrypsin- hydrolysis of peptide backbone

Incorporates covalent catalysis and acid-base catalysis(details next lecture)

Carbonic Anhydrase- equilibrate carbon dioxide and carbonic acid

Metal ion catalysis(near catalytic perfection)

Restriction Enzymes- sequence-specific cleavage of DNA backbone

hydrolysis via covalent catalysis(extreme specificity)

NMP Kinases- transfer of phosphate group between 2 substrates

group transfer using metal ion catalysis(very efficient- avoid loss of the phosphate group)

Reactions Proceed in a Series of Steps

S PNet Reaction:

SubstrateBinding

ChemicalRearrangement

ProductRelease

E + S ES EP E + P

Each of these steps might be analysed further to understand atomic details.

Enzyme Catalysed Pathway:

A + B P + Q

Group Transfer Reactions Have 2 Substrates

S1-G + S2 S1’ + S2’-G

2 Substrates 2 Products

Substrates and Products can be bound and released in various orders:

Sequential Displacement:Ordered SequentialRandom Sequential

Double Displacement

Net Reaction:

Ordered Sequential Reactions

All substrates bind before any product is released.

SubstrateBindingSteps

ProductReleaseSteps

ChemicalRearrangementSteps

(protons tranferred)

(e.g. lactate dehydrogenase)

Random Sequential Reaction

Order of substrate binding and product release is random.

SubstrateBindingSteps

ProductReleaseSteps

ChemicalRearrangementSteps

(phospatetranferred)

(e.g. creatine kinase)

Double Displacement (or Ping-Pong) Reaction

Some products released before all substrates are bound.

SubstrateBindingStep

ChemicalRearrangementStep

ProductReleaseStep

ChemicalRearrangementStep

SubstrateBindingStep

ProductReleaseStep

(amino group tranferred)

(amino groupon enzyme)

(e.g. aspartate aminotransferase)

Summary:

The chemical reactions catalyzed by most enzymes can be classifiedinto one of 6 general types of reactions.

A few catalytic strategies are used by most enzymes regardless ofthe particular chemistry they perform.

Enzyme-catalyzed reactions proceed in an organized series of stepseach of which can be considered separately.

Key Concepts:Covalent catalysisNucleophileAcid-Base CatalysisMetal-ion CatalysisCatalysis by Approximation

Electrostatic Effects, Desolvation, Induced Fit

Displacement reactions