Enzymes catalysis Enzyme catalyzed chemical reactions via both non-covalent and covalent interactions Transient chemical reactions (i.e., covalent interactions) often occur between substrates and functional groups in the active sites of enzymes, thus providing an alternative reaction path. Noncovalent interactions between substrates and enzymes will generate a binding energy (ΔGB), which will lower the activation energy of the reactions.

Weak interactions between enzyme and substrate are optimized in the transition state Enzyme and substrate was proposed to complement each other like “ a lock and the key” (Emil Fischer, 1894). Years later, it was realized that an enzyme completely complementary to its substrate would be a very poor enzyme! According to the transition state theory, an enzyme must be complementary to the reaction transition state of the reactant (Haldane, 1930; Pauling, 1946).

An effective enzyme must have its active site complementary to the transition state of the reaction. Activation energy increases! ES E-transition state E + P

The transition state theory of enzyme catalysis has strong supporting evidences The idea of transition-state analogs was suggested in accordance with the transition state theory (Pauling, 1940s) and were later proved to be correct: such analogs bind to enzymes 102 to 106 times more tightly than normal substrates. The idea of catalytic antibodies was also suggested by this theory (Jencks, 1969) and was proved to be correct later (Lerner and Schultz, 1980s).

Transition-state analogs can be designed according to the proposed reaction mechanism and used as antigens for making catalytic antibodies. Catalytic antibodies Transition-state analog Transition-state Catalytic antibodies Transition-state analog

The binding energy made available by the noncovalent enzyme-substrate interactions often provide a major driving force for enzyme catalysis.

Enzyme catalysis I. Rate Acceleration Enzyme accelerate the rate of reaction: In most case, initial interaction is noncovalent (ES) making use of hydrogen bonding, electrostatic, hyodrophobic and vander Waals force to effect binding. ES: Catalytic groups are now an integral part of the same molecule, the reaction of enzyme bound substrates will follow first order rather than second order kinetics. The classic way that an enzyme increases the rate of a bimolecular reaction is to use binding energy to simply bring the two reactants in close proximity. •If ΔG‡ is the change in free energy between the ground state and the transition state, then ΔG‡=ΔH‡–tΔS‡. In solution chemistry, the transition state would besignificantly more ordered than the ground state, and ΔS‡ would therefore benegative, making ΔG‡ more positive, or less favorable. •The formation of a transition state is accompanied by losses in translationalentropy as well as rotational entropy. Enzymatic reactions take place within the confines of the enzyme active-site wherein the substrate and catalytic groups on the enzyme act as one molecule. Therefore, there is no loss intranslational or rotational energy in going to the transition state. E + S --> ES --> [EX*] --> EP --> E + P

Enzyme catalysis II. Binding Energy in Catalysis: Favorable interaction between the enzyme and substrate result in a favorable intrinsic binding energy. Entropy is lost when substrate binds to the enzyme. Two entities become one. Substrate is less able to rotate. Substrate become more ordered. Weak interactions between the enzyme and substrate are optimize and stabilize the transition state. E + S --> ES --> [EX*] --> EP --> E + P (weak) (stronger)

Factors involved in rate acceleration Desolvation: When substrate binds to the enzyme surrounding water in solution is replaced by the enzyme. This makes the substrate more reactive by destablizing the charge on the substrate. Expose a water charged group on the substrate for interaction with the enzyme. Also lowers the entropy of the substrate (more ordered).

Factors involved in rate acceleration……. Strain and Distortion: When substrate bind to the enzyme, it may induces a conformational change in the active site to fit to a transition state. Frequently, in the transition state, the substrate and the enzyme have slightly different structure (strain or distortion) and increase the reactivity of the substrate. cyclic phosphate ester Acylic phospodiester

Binding energy can be used for selecting specific substrates and overcome the ΔG ‡ The reduction in entropy of oriented substrates. Desolvation of the substrates. Distortion of substrates for converting to the transition state. Proper alignment of catalytic function groups via induced fit (conformational change) in the enzyme active site. The consumption of binding energy in such processes will help lower the ΔG ‡ , thus increasing the reaction rate.

Catalytic Strategies Catalysis by approximation Covalent catalysis In reactions that include two substrates, the rate is enhanced by bringing the two substrates together in a proper oirentation. Covalent catalysis The active site contains a reactive group, usually a powerful nucleophile that become temporarily covalently modified in the course of catalysis. General acid-base catalysis A molecule other than water plays the role of a proton donor or acceptor. Metal ion catalysis Metal ions can serve as electrophilic catalyst, stabilizing negative charge on a reaction intermediate.

Catalytic Strategies Approximation Enzyme serves as a template to bind the substrates so that they are close to each other in the reaction center. - Bring substrate into contact with catalytic groups or other substrates. - Correct orientation for bond formation. - Freeze translational and rotational motion.

Catalytic Strategies Approximation Bimolecular reaction (high activation energy, low rate). Unimolecular reaction, rate enhanced by factor of 105 due to increased probability of collision/reaction of the 2 groups Constraint of structure to orient groups better (elimination of freedom of rotation around bonds between reactive groups), rate enhanced by another factor of 103, for 108 total rate enhancement over bimolecular reaction

Catalytic Strategies Covalent catalysis The principle advantage of using an active site residue instead of water directly is that formation of covalent linkage leads to unimolecular reaction, which is entropically favored over the bimolecular reaction. Enzyme that utilize covalent catalysis are generally two step process: formation and breakdown of covalent intermediate rather than catalysis of the single reaction directly. Y should be a better leaving group than X. X is a better attacking group then Z. Covalent intermediate should be more reactive than substrate.

Catalytic Strategies Covalent catalysis Phosphoramidate Intermediate ATP-Dependent DNA Ligase O P H 2 C OH N NH Lys + ATP LigaseﾐAdenylate Phosphoramidate Intermediate Lys N P O Nucleoside H +

Catalytic Strategies Covalent catalysis What kind of groups in proteins are good nucleophiles: Aspartate caboxylates Glutamates caboxylates Cystine thiol- Serine hydroxyl- Tyrosine hydroxyl- Lysine amino- Histadine imidazolyl-

Catalytic Strategies Acid-base catalysis A proton (H+) is transferred in the transition state. Specific acid-base catalysis: Protons from hydronium ion (H3O+) and hydroxide ions (OH-) act directly as the acid and base group. General acid-base catalysis: Catalytic group participates in proton transfer stabilize the transition state of the chemical reaction. Protons from amino acid side chains, cofactors, organic substrates act as Bronsted-Lowry acid and base group.

Catalytic Strategies Acid-base catalysis Transition State of Stabilization by a General Acid (A) or General Base (B) in Ester Hydrolysis by Water. Transition state can be stabilized by acid group (A-H) acting as a partial proton donor for carbonyl oxygen of the ester - Enhance the stability of partial negative charge on the ester. Alternatively, enzyme can stabilize transition state by basic group (B:) acting as proton acceptor. For even greater catalysis, enzyme can utilize acid and base simultaneously This type of Acid-base is common mechanism of transition state stabilization in enzymatic reaction.

Catalytic Strategies Acid-base catalysis Histidine pKa is around 7. It is the most effective general acid or base. Example: RNase A: His 12 General Base Abstracts a proton from 2’ hydroxyl of 3’ nucleotide. His 119 General acid Donates a proton to 5’ hydroxyl of nucleoside. This type of Acid-base is common mechanism of transition state stabilization in enzymatic reaction.

Catalytic Strategies Acid-base catalysis Histidine pKa is around 7. It is the most effective general acid or base. Example: RNase A: His 12 General Base Abstracts a proton from 2’ hydroxyl of 3’ nucleotide. His 119 General acid Donates a proton to 5’ hydroxyl of nucleoside. This type of Acid-base is common mechanism of transition state stabilization in enzymatic reaction. 2’-3’ cyclic phosphate intermediate Net Proton Transfer from His119 to His12

Acid and base roles are reversed for H12 and H119 Catalytic Strategies Acid-base catalysis Histidine pKa is around 7. It is the most effective general acid or base. Example: RNase A: His 12 General Base Abstracts a proton from 2’ hydroxyl of 3’ nucleotide. His 119 General acid Donates a proton to 5’ hydroxyl of nucleoside. This type of Acid-base is common mechanism of transition state stabilization in enzymatic reaction. Water replaces the released nucleoside Acid and base roles are reversed for H12 and H119

Original Histidine protonation states are restored Catalytic Strategies Acid-base catalysis Histidine pKa is around 7. It is the most effective general acid or base. Example: RNase A: His 12 General Base Abstracts a proton from 2’ hydroxyl of 3’ nucleotide. His 119 General acid Donates a proton to 5’ hydroxyl of nucleoside. This type of Acid-base is common mechanism of transition state stabilization in enzymatic reaction. Original Histidine protonation states are restored

General Acids

General Bases

Catalytic Strategies Metal ion catalysis. Metal ions can … Electrostatically stabilizing or shielding negative charges. Act to bridge a substrate and nucleophilic group. Bind to substrates to insure proper orientation. Participate in oxidation/reduction mechanisms through change of oxidation state. Shield negative charges on substrate group that will otherwise repaile attack of nucleophile (i.e: stabilizing negative charge on a reaction intermediate). Increase the reactivity of a group by electron withdraw or by altering pKa (i.e: Generate a nucleophile by increasing the acidity of a nearby molecule). Act to bridge a substrate and nucleophilic group.

Catalytic Strategies Metal ion catalysis. Can stabilize developing negative charge on a leaving group, making it a better leaving group. Shield negative charges on substrate group that will otherwise repaile attack of nucleophile (i.e: stabilizing negative charge on a reaction intermediate). Increase the reactivity of a group by electron withdraw or by altering pKa (i.e: Generate a nucleophile by increasing the acidity of a nearby molecule). Act to bridge a substrate and nucleophilic group.

Catalytic Strategies Metal ion catalysis. Can stabilize developing negative charge on a leaving group, making it a better leaving group. Can shield negative charges on substrate group that will otherwise repel attack of nucleophile. Shield negative charges on substrate group that will otherwise repaile attack of nucleophile (i.e: stabilizing negative charge on a reaction intermediate). Increase the reactivity of a group by electron withdraw or by altering pKa (i.e: Generate a nucleophile by increasing the acidity of a nearby molecule). Act to bridge a substrate and nucleophilic group.

Catalytic Strategies Metal ion catalysis. Can stabilize developing negative charge on a leaving group, making it a better leaving group. Can shield negative charges on substrate group that will otherwise repaile attack of nucleophile. Can increase the rate of a hydrolysis reaction by forming a complex with water, thereby increasing water’s acidity. Shield negative charges on substrate group that will otherwise repaile attack of nucleophile (i.e: stabilizing negative charge on a reaction intermediate). Increase the reactivity of a group by electron withdraw or by altering pKa (i.e: Generate a nucleophile by increasing the acidity of a nearby molecule). Act to bridge a substrate and nucleophilic group.

Specific Forces Involved in Enzyme-Substrate Complex Formation 1. Covalent Bond 2. Ionic (or Electrostatic) Interactions 3. Ion-Dipole and Dipole-Dipole Interactions 4. Hydrogen Bonds 5. Charge Transfer Complexes 6. Hydrophobic Interactions 7. Vander Waals Forces