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How is TS Stabilization Achieved?

acid-base catalysis: give and take protons covalent catalysis: change reaction paths metal ion catalysis: use redox cofactors, pKa shifters electrostatic catalysis: preferential interactions with TS

Acid-base Catalysis: Chemical Example


Consider ester hydrolysis:
O R O CH3 O O O H O
+

H -

+ H
CH 3

R O H

C 3 H O

Water is a poor nucleophile, and methanol is a poor leaving group Aqueous hydrolysis can be catalyzed either by acids or by bases Enzymes can do acid and base catalysis simultaneously

General Acid-Base Catalysis


Example: amide hydrolysis

Amino Acids in General Acid-Base catalysis

Covalent Catalysis: Chemical Example


O H3C
O H3C O O CH3 fast O + N CH3 O

O O CH3

H2O slow H3C

H3C

+ + 2H O

H3C

.. N ..
H O H

The anhydride hydrolysis reaction is catalyzed by pyridine, a better nucleophile than water (pKa=5.5). Hydrolysis is accelerated because of charge loss in the transition state makes pyridine a good leaving group.

CH3 OH

.. N

+ HC 3

O
H
+

+ N

Covalent Catalysis: In Enzymes


Proteases and peptidases chymotrypsin, elastase, subtilisin reactive serine nucleophile Some aldehyde dehydrogenase glyceraldehyde-3phosphate dehydrogenase reactive thiolate nucleophile Aldolases and decarboxylases amine nucleophile Dehalogenases carboxylate nucleophile
NH2

HO

O S

Chymotrypsin Mechanism
Step 1: Substrate Binding Endo-peptidase specificity
P3 - P2 - P1-|-P1- P2- P3

Chymotrypsin Mechanism
Step 2: Nucleophilic Attack Serine protease catalytic triad activates serine hydroxyl

Chymotrypsin Mechanism
Step 3: Substrate Cleavage N-terminal peptide released Covalent acyl intermediate

Chymotrypsin Mechanism
Step 4:Water attack

Chymotrypsin Mechanism
Step 5: De-acylation

Chymotrypsin Mechanism
Step 6: Serine hydroxyl restored

Chymotrypsin Mechanism
Step 7: Product Dissociates

Peptidoglycan and Lysozyme

Peptidoglycan is a polysaccharide found in many bacterial cell walls Cleavage of the cell wall leads to the lysis of bacteria Lysozyme is an antibacterial enzyme

General Acid-Base + Covalent Catalysis: Cleavage of Peptidoglycan by Lysozyme


X-ray structures of lysozyme with bound substrate analogs show that the C-1 carbon is located between Glu 35 and Asp 52 residues.

Cleavage of Peptidoglycan by Lysozyme: Two Successive SN2 Steps Model


Asp 52 acts as a nucleophile to attack the anomeric carbon in the first SN2 step
Glu 35 acts as a general acid and protonates the leaving group in the transition state. Water hydrolyzes the covalent glycosyl-enzyme intermediate

Glu 35 acts as a general base to deprotonate water in the second SN2 step

Enzyme Regulation
Allosteric regulation,
heterotropic ligand binding modulates substrate binding and catalysis, Feedback regulates metabolic pathways

Homotropic regulation Multisubunit Covalent modification Reversible


Phosphorylation, nucleotides, lipid anchors

Proteolysis converts inactive pro-enzymes (zymogens) to active

Allosteric Regulation; ATCase

Chapter 6: Summary
In this chapter, we learned about:
why nature needs enzyme catalysis how enzymes can accelerate chemical reactions how chymotrypsin breaks down peptide bonds how to perform and analyze kinetic studies how to characterize enzyme inhibitors