Chymotrypsin Lecture. Aims: to understand (1) the catalytic strategies used by enzymes and (2) the mechanism of chymotrypsin

Transcription

1 Chymotrypsin Lecture Aims: to understand (1) the catalytic strategies used by enzymes and (2) the mechanism of chymotrypsin

2 What s so great about enzymes? They accomplish large rate accelerations ( fold) in an aqueous environment using amino acid side chains and cofactors with limited intrinsic reactivity They are exquisitely specific

3 Chymotrypsin Digestive enzyme secreted by the pancreas Serine protease Large hydrophobic amino acids Specific for the peptide carbonyl supplied by an aromatic residue (eg Tyr, Met)

4 Specificity of chymotrypsin Nucleophilic attack Carbonyl bond Hydrophobic amino acids

5 Common catalytic strategies 1. Covalent catalysis Reactive group (nucleophile) Hydroxide ion 2. General acid-base catalysis proton donor/acceptor (not water) 3. Metal-ion catalysis Nucleophile or electrophile eg Zn Form bridge between enzyme and substrate 4. Catalysis by approximation Two substrates along a single binding surface or, combination of these strategies eg an example of use of 1 & 2 is chymotrypsin

6 Proteases Catalyse a Fundamentally Difficult Reaction They cleave proteins by hydrolysis the addition of water to a peptide bond

7 Half life for hydrolysis of typical peptide is years. Chymotrypsin accelerates the rate of cleavage to 100 s -1 (>10 12 enhancement). Resonance structure The carbon-nitrogen bond is strengthened by its double-bond character carbonyl carbon atom is less electrophilic less susceptible to nucleophilic attack Enzyme must facilitate nucleophilic attack on normally unreactive carbonyl group

8 Identification of the reactive serine Around 1949 the nerve gas di-isopropyl-fluorophosphate was shown to inactivate chymotrypsin 32 P-labelled DIPF covalently attached to the enzyme When labelled enzyme was acid hydrolysed the phosphorus stuck tightly; the radioactive fragment was O- phosphoserine Sequencing established the serine to be Ser195 Among 28 serines, Ser195 is highly reactive, why?

9 An unusually reactive serine in chymotrypsin

10 Probing enzyme mechanism Colourless Carboxylic acid Catalysed by chymotrypsin Yellow product Measure absorbance

11 Kinetics of chymotrypsin catalysis

12 Covalent catalysis Two stages

13 Stage 1- acylation (p-nitrophenolate)

14 Deacylation through hydrolysis Covalent bond Carboxylic acid

15 Location of the active site in chymotrypsin Hydrogen bonded His 57 Asp chains Catalytic Triad

16 The catalytic triad Nucleophile Arrangement polarises serine hydroxyl group Histidine becomes a proton acceptor Stabilised by Aspartate

17 Peptide hydrolysis by chymotrypsin

18 Step 1 substrate binding Nucleophilic attack

19 2. Formation of the tetrahedral intermediate Ser 195 -ve charge on oxygen stabilised

20 3. Tetrahedral intermediate collapse Generates acyl-enzyme Transfer of His proton amine component formed

21 4.Release of amine component (acylation of enzyme)

22 5. Hydrolysis (deacylation)

23 6. Formation of tetrahedral intermediate Histidine draws proton from water Hydroxyl ion attacks carbonyl

24 7. Formation of carboxylic acid product

25 8. Release of carboxylic acid

26 NH groups (O 2 ) Stabilisation of intermediates

27 WHY DOES CHYMOTRYPSIN PREFER PEPTIDE BONDS JUST PAST RESIDUES WITH LARGE HYDROPHOBIC SIDE CHAINS?

28 Specificity of chymotrypsin Nucleophilic attack Hydrophobic amino acids

29 Specificity pocket of chymotrypsin (S1-pocket) Pocket Lined with hydrophobic residues Substrate side chain binding phenylalanine S1-subsite

30 Specificity nomenclature for protease substrate interactions. N-terminal Scissile bond C-terminal More complex specificity P potential sites of interaction with the enzyme (P carboxyl side) S Corresponding binding site on the enzyme (specificity pocket)

31 S1 pockets confer substrate specificity Arg,lys (+ve charge) Ala, ser (small side chain)

32 Subtilisin cf Chymotrypsin Catalytic triad

33 Site directed mutagenesis K M unchanged

34 Not all proteases utilise serine to generate nucleophile attack

35 Proteases and their active sites 1.

36 Proteases and their active sites 2.

37 Proteases and their active sites 3.

38 Activation strategy 1. His Cys Nucleophile Eg Papain

39 Eg Renin Activation strategy 2. Nucleophile Asp Asp

40 Activation strategy 3. Nucleophile Eg carboxypeptidase A

41 Active site acts to :- Activation strategy a) Activate a water molecule or other nucleophile (cys, ser) b) Polarise the peptide carbonyl c) Stabilise a tetrahedral intermediate.

42 Protease inhibitors are important drugs

43 HIV protease Dimeric aspartyl protease Cleaves viral proteins activation Aspartate residues

44 HIV protease inhibitor symmetry

45 HIV protease-indovir complex Asp

46 Berg Tymoczko Stryer Biochemistry Sixth Edition Chapter 9: Catalytic Strategies Copyright 2007 by W. H. Freeman and Company