Advanced Bioprocess Engineering Enzymes & Enzymes Kinetics Lecturer Dr. Kamal E. M. Elkahlout Assistant Prof. of Biotechnology

III. Other 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). Enzyme (Basic Principle)

III. Other 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. Enzyme (Basic Principle) Rate: cyclic phosphate esterAcylic phospodiester

Catalytic Strategies Catalysis by approximation –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 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. Approximation

a)Bimolecular reaction (high activation energy, low rate). b)Unimolecular reaction, rate enhanced by factor of 10 5 due to increased probability of collision/reaction of the 2 groups c)Constraint of structure to orient groups better (elimination of freedom of rotation around bonds between reactive groups), rate enhanced by another factor of 10 3, for 10 8 total rate enhancement over bimolecular reaction Approximation 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.

Catalytic Strategies Covalent catalysis 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. 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.

Catalytic Strategies Covalent catalysis ATP-Dependent DNA Ligase Phosphoramidate Intermediate LysNPONucleoside O O H H +

Catalytic Strategies Covalent catalysis What kind of groups in proteins are good nucleophiles: Aspartate caboxylates Glutamates caboxylates Cystinethiol- Serinehydroxyl- Tyrosinehydroxyl- Lysineamino- Histadineimidazolyl-

Catalytic Strategies Covalent catalysis Schiff Base Formation A Schiff base may form from the condensation of an amine with a carbonyl compound. The Schiff base (protonated at neutral pH) acts as an electron sink that greatly stabilizes negative charge that develops on the adjacent carbon. Stable Intermediate

Catalytic Strategies Covalent catalysis Schiff Base Formation Enzymes that form Schiff base intermediates are typically irreversibly inhibited by the addition of sodium borohydride (Na + BH 4 – ). Borohydride reduces the Schiff base and “traps” the intermediate such that it can no longer be hydrolyzed to release the product from the enzyme. This is often used as evidence for a mechanism involving an enzyme-linked Schiff base intermediate.

Catalytic Strategies Acid-base catalysis A proton (H + ) is transferred in the transition state. Specific acid-base catalysis: Protons from hydronium ion (H 3 O + ) and hydroxide ions (OH - ) act directly as the acid and base group. General acid-base catalysis: Catalytic group participates in protein 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

Catalytic Strategies Acid-base catalysis Histidine pK a 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.

Catalytic Strategies Acid-base catalysis Histidine pK a 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. 2’-3’ cyclic phosphate intermediate Net Proton Transfer from His119 to His12

Catalytic Strategies Acid-base catalysis Histidine pK a 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. Water replaces the released nucleoside Acid and base roles are reversed for H12 and H119

Catalytic Strategies Acid-base catalysis Histidine pK a 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. Original Histidine protonation states are restored

Catalytic Strategies Metal ion catalysis. Metal ions can … Electrostatically stabilizing or shielding negative charges. Act much like a proton but can be present in high concentration at neutral pH and can have multiple positive 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.

Catalytic Strategies Metal ion catalysis. Metal ions can … Electrostatically stabilizing or shielding negative charges. Act much like a proton but can be present in high concentration at neutral pH and can have multiple positive 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.

Catalytic Strategies Metal ion catalysis. 1)Can stabilize developing negative charge on a leaving group, making it a better leaving group.

Catalytic Strategies Metal ion catalysis. 1)Can stabilize developing negative charge on a leaving group, making it a better leaving group. 2)Can shield negative charges on substrate group that will otherwise repel attack of nucleophile.

Catalytic Strategies Metal ion catalysis. 1)Can stabilize developing negative charge on a leaving group, making it a better leaving group. 2)Can shield negative charges on substrate group that will otherwise repaile attack of nucleophile. 3)Can increase the rate of a hydrolysis reaction by forming a complex with water, thereby increasing water’s acidity.

Catalytic Strategies Metal ion catalysis. Examples: