Hypothetical substrate docking in enzyme’s active site. Substrate is geometrically and electronically compatible with active site. Enzymes are also stereospecific.

ES complex formation

How is  G ‡ lowered? Many enzymes stabilize the transition state in addition to the mechanisms described below through relatively tight binding. Mechanisms of catalysis 1. Acid-Base catalysis 2. Covalent catalysis 3. Metal ion catalysis 4. Proximity and orientation effects 5. Electrostatic catalysis

1. Acid-Base catalysis (H + transfer between E and S) Acid catalysis

Base catalysis

2. Covalent catalysis (covalent bond between E and S) Amine in enzyme reacts with carbonyl group to form Schiff base Elimination of CO 2 then does not lead to an unstable transition state

Schiff base breaks down to amine catalyst and acetone 3. Metal ion catalysis (metal ions can mediate redox reactions or electrostatically promote reactions).

Proximity/Orientation No catalyst: slow b/c few encounters Catalyst: faster

5. Electrostatic catalysis Most substrate molecules have a hydration shell that must be removed to allow reaction. Enzyme active sites are often non-aqueous (not necessarily non-polar) and they require that water is removed from the substrate before binding can occur. The non-aqueous active site allows stronger electrostatic interactions.

Chymotrypsin. 241 amino acid protein with 2 domains and an active site with 3 residues important for catalysis shown in red (His 57, Asp 102 and Ser 195). Chymotrypsin Digestive enzyme synthesized in pancreas and secreted to small intestine. Has broader specificity than most enzymes. Hydrolyzes the peptide bond following large nonpolar residues like Phenylalanine, Tryptophan or Tyrosine. Leu-Ala-Tyr-Ile-Asp

Asp 102 His 57 Ser 195 Chymotrypsin is part of a class of enzymes known as Serine Proteases. Uses acid-base and covalent catalysis, and it stabilizes the transition state.

16 Let’s look at the detailed reaction mechanism for chymotrypsin, a protease: It hydrolyzes peptide bonds immediately C-terminal to aromatic amino acids With a rate enhancement of > 10 9 And illustrates several principles Transition state stabilization Acid-base catalysis Covalent catalysis A two-phase reaction Leu-Ala-Tyr-Ile-Asp

17 Appreciate the contribution of protein folding PDB: 7GCH

18 Mechanism in the Substrate Pocket Phase I - acylation of Ser 195 Phase 2 – deacylation of Ser 195 Follow the roles of the “catalytic triad” through the reaction: Ser 195, His 57 and Asp 102 What is the role of Gly 193 ?

19 In the active site of chymotrypsin, know the chemical roles of: Histidine 57 Aspartate 102 Serine 195 Glycine 193

20 Chymotrypsin Mechanism -1

21 Chymotrypsin Mechanism -2

22 Chymotrypsin Mechanism - 3

23 Chymotrypsin Mechanism - 4

24 Chymotrypsin Mechanism - 5

25 Chymotrypsin Mechanism - 6

26 Chymotrypsin Mechanism - 7

Asp102 His57 Ser195 Catalytic Triad H H Chymotrypsin Catalytic Mechanism A1 N C C N [HOOC] H O C C N C C [NH 2 ]C O Check substrate specificity

Asp102 His57 Ser195 H H Chymotrypsin Catalytic Mechanism A2 N C C N [HOOC] H O C C N C C [NH 2 ] C O First Transition State

H H Chymotrypsin Catalytic Mechanism A3 N C C N [HOOC] H O C C N C C [NH 2 ] C O Acyl-Enzyme Intermediate

H Chymotrypsin Catalytic Mechanism D1 N-H C C N [HOOC] H O C C N C C [NH 2 ] C O H O H Acyl-Enzyme Water Intermediate

H Chymotrypsin Catalytic Mechanism D2 O O C C N C C [NH 2 ] C H Second Transition State O H

H Chymotrypsin Catalytic Mechanism D3 O C C N C C [NH 2 ] C O O H Deacylation H