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 from enzyme to enzyme, similar strategies are used to carry them out.

General Acid-Base Catalysis: Common Types of Catalysis Covalent Catalysis: A group on the enzyme becomes covalently modified during reaction, e.g. by forming a covalent bond to the substrate during the reaction. General Acid-Base Catalysis: A group on the enzyme acts as an acid or base: it removes a proton 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 chemical rearrangement or binding step. Catalysis by Approximation: The enzyme holds two substrates near in space and in precisely the correct spatial orientation to optimize their reaction. (Most enzymes use a combination of several of these strategies)

Rate acceleration by transient formation of a Covalent Catalysis Rate acceleration by transient formation of a COVALENT enzyme-substrate bond Enzyme Substrate Covalent Intermediate Products 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. Covalent Catalysis Requires a Highly Reactive Group Some chemical group that can invade the substrate- usually a nucleophile. Nucleophile: an electron-rich group that attacks nuclei. Examples of nucleophiles among protein functional groups: unprotonated His imidazole unprotonated a-amino group unprotonated sidechain amino group of Lys thiolate anion (-S-) of Cys aliphatic -OH of Ser sidechain carboxylates of Glu, Asp (Also some coenzymes.)

General Acid-Base Catalysis Specific functional groups in enzyme structure are positioned to either Donate 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 to then accept a proton (act as a general base) later in catalytic mechanism for catalyst to be regenerated in original form. Examples of general acid/base catalysts among protein functional groups: His imidazole a-amino group thiol of Cys R group carboxyls of Glu, Asp Sidechain amino group of Lys Aromatic OH of Tyr Guanidino group of Arg

Metal Ion Catalysis Metal ions can be used in a variety of ways by enzymes. (In fact they are so useful 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 charge state (eg. iron Fe2+ and Fe3+) can gain or lose electrons during the reaction, avoiding unstable charged intermediates. Shielding or stabilizing negative charges: If charge on substrate or on transition state is an integral part of the reaction, the enzyme can form strong ionic interactions with that charge which are stabilizing.

- + Enzyme Substrate +2 Stable Unstable +2 +3 Binding and orientation of substrate AND/OR Shielding or stabilizing negative charges Stable Unstable +2 + +3 Redox Reactions

Catalysis by Approximation Proximity: Reaction between bound molecules doesn't require an improbable collision of 2 molecules. They're already in "contact" (increases local concentration of reactants). Orientation: Reactants are not only near each other on enzyme, they're oriented in optimal position to react. The improbability of colliding in correct orientation is taken care of. Substrates held close in space and in correct orientation Results in more efficient reaction pathway

Other Effects that Stabilize the Transition State Electrostatic Effects: Increase in strength of ionic interactions due to lower dielectric constant. Desolvation: Exclusion of water from active site. Induced Fit: Change in conformation of enzyme or substrate to optimize interactions.

Electrostatic Effects Enhancement of the attraction between opposite charges by various means. Coulomb’s Law: Examples: Providing a lower dielectric constant of the environment in the active site (hydrophobic environment) Altering pK values of specific functional groups. Stabilizing a particular conformation of the critical groups in the active site by electrostatic interactions. Stabilizing (binding) a charged intermediate or transition state by providing an oppositely charged enzyme group close by.

Desolvation By sequestering reactants away from water, two major effects are Achieved: 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.

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

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)

S P E + S ES EP E + P Reactions Proceed in a Series of Steps Substrate Net Reaction: Enzyme Catalysed Pathway: Substrate Binding Chemical Rearrangement Product Release E + S ES EP E + P Each of these steps might be analysed further to understand atomic details.

Group Transfer Reactions Have 2 Substrates S1-G + S2 S1’ + S2’-G A + B P + Q Net Reaction: 2 Substrates 2 Products Substrates and Products can be bound and released in various orders: Sequential Displacement: Ordered Sequential Random Sequential Double Displacement

Ordered Sequential Reactions (e.g. lactate dehydrogenase) All substrates bind before any product is released. (protons tranferred) Substrate Binding Steps Chemical Rearrangement Steps Product Release Steps

Random Sequential Reaction (e.g. creatine kinase) Order of substrate binding and product release is random. (phospate tranferred) Substrate Binding Steps Chemical Rearrangement Steps Product Release Steps

Double Displacement (or Ping-Pong) Reaction (e.g. aspartate aminotransferase) Some products released before all substrates are bound. (amino group tranferred) Substrate Binding Step Product Release Step Substrate Binding Step Product Release Step Chemical Rearrangement Step (amino group on enzyme) Chemical Rearrangement Step

Summary: The chemical reactions catalyzed by most enzymes can be classified into one of 6 general types of reactions. A few catalytic strategies are used by most enzymes regardless of the particular chemistry they perform. Enzyme-catalyzed reactions proceed in an organized series of steps each of which can be considered separately. Key Concepts: Covalent catalysis Nucleophile Acid-Base Catalysis Metal-ion Catalysis Catalysis by Approximation Electrostatic Effects, Desolvation, Induced Fit Displacement reactions