Metal ions as Covalent Catalysts- Carbonic anydrase

HCO3-CO2 H2O+ + H+

Metal ion covalent catalysis- Carbonic anydrase

HCO3-CO2 H2O+ + H+

Proximity Effects of Intramolecular CatalysisProximity Effect-is defined as a rate increase due to two reactants being brought out of a dilute environment and placed closer together (huge loss of entropy without enzyme)

enzymes do this by providing a docking site and a micro environment allowing proper substrate orientation for reaction

Contributes to the loss of the substrates freedom of movement (loss of entropy pg 72) Binding energy overcomes loss of entropy

Increases chances of reaction --molecules are closer together on as ES complex (becomes an intramolecular process) – increase in effective concentration of catalytic groups

Proximity Effect accounts for only part of rate enhancement

Desolvating the substrate from water (or other solvent) eliminates energy barrier imposed by ordered solvent molecules (get some entropy back) – accelerating reaction (electrostatic catalysis?) (mutation of catalytic residues-not completely dead)

Next

PLP catalysis

Nucleophilic catalysis

Microscopic reversibility and kinetic equivalence

Previous lecture:

•Chemical catalysis-stabilization of transition state

•Acid-base catalysis, Electrophilic catalysis, Covalent catalysis, Metal ion catalysis

•Importance of pKa values

•Proximity effect and desolvation (creation of microenvironment)

Today:•PLP – mediated catalysis

•Nucleophilic catalysis

•Microscopic reversibility

Coenzyme CatalysisCoenzyme (Cofactor)

small organic molecule or metal ion essential to the catalytic action of an enzyme

Common cofactors:FMN flavin mononucleotideFAD flavin adenine dinucleotideNAD+/NADH nicotinamide adenine dinucleotideNADP+ / NADPH nicotinamide adenine dinucleotide phosphatePLP pyridoxal 5’-phosphatePMP pyridoxamine 5’-phosphateTHF tetrahydrofolateCoASH coenzyme AGSH glutathioneATP adenosine triphosphate

Pyridoxal Phosphate (PLP) Enzymology

•PLP is the modified form of Vitamin B6 (pyridoxal)

•PLP is a coenzyme heavily involved in amino acid transformation reactions

•Central to its mechanism is the formation of a Schiff base with the enzyme (through a covalent bond with Lys)

•Schiff base is formed through the condensation of an amine with a carbonyl group.

•Serves to stabilize carbanion intermediates of amino acids

Pyridoxal Phosphate (PLP) Enzymology

PLP most commonly involved in decarboxylation or transaminationreactions at the α-Carbon – Also, elimination, and replacementreactions at the β-Carbon

N

HOOH

Me

HO

vitamin B6

N

OOH

Me

O

PLP

H

PO

O

O

Pyridoxal Phosphate (PLP) Enzymology

Pyridoxal Phosphate (PLP) Enzymology

•PLP-requiring enzymes act via the formation of a planar Schiff base between the amino acid to be modified(substrate) and the coenzyme

Pyridoxal Phosphate (PLP) Enzymology

•PLP-requiring enzymes act via the formation of a planar Schiff base between the amino acid to be modified (substrate) and the coenzyme

•That leads to the formation of a resonance-stabilized carbanion with a quinoidstructure

•The key to catalysis is the electrophilic nitrogen of the pyridine ring - draws electrons away from the substrate and stabilizes the carbanionintermediate (electron sink)

Aspartate Aminotransferase active site with PLP

Dr. Jack Kirsch-Berkeley

Nucleophilic Catalysis

Nucleophilic Catalysis

Catalytic residues vs water

•Water is less nucleophilic than hydroxyls and thiols

•Intramolecular reaction vs Intermolecular (favoured entropically)

•Catalytic residues are more structured in place vs water molecules

Nucleophilic attack on carbonyls

•Rate increases with increasing electron withdrawing substituents

•Rate increases with increasing electron withdrawing leaving groups

•Rate increases with increasing strength of base

•While acid-base catalysis depends on pKa’s alone, nucleophiliccatalysis depends on chemical nature of reactants and microenvironment of enzyme active site

Nucleophilic Catalysis

On Hard Groups

•Includes attack on an ester, carbonyl, or amide

•Formation of stable intermediate (often isolated)

•Carbon valence remains four, phosphate ester valence remains five

•Central feature is the strength of the nucleophilic base

•Amines (K,H), thiolate anions (C), are more nucleophilic than oxyanions (S,T,Y)

Nucleophilic Catalysis

On Soft groups

•Includes attack on saturated carbons

•Formation of abnormal transition states (in terms of valence)

•Central feature is polarizability of nucleophile in addition to basic strength

•thiolate anions (C) are reactive in these situations

•Leaving group activation – neutral groups are more readily displaced than highly basic groups

S-adenosyl methionine

Principle of microscopic reversibility: the forward and reverse of a particular reaction are related as mirror images. Thus, each mechanistic step must be at equilibrium when the whole system is at equilibrium

•Every reaction is reversible•The sequence and structure of transition states and reactive intermediates in the mechanism of a reversible reaction must be the same, but in reverse order•The lowest-energy pathway in the forward direction will be the lowest-energy pathway in the reverse direction.

Under the same set of conditions:

Next Class

Kinetic Isotope Effects

Steady state kinetics