BB12 – Chymotrypsin and proteases

Question 0

Common catalytic strategies

Answer 0

1. covalent catalysis
2. general acid-base catalysis
3. metal-ion catalysis
4. catalysis by approximation

Question 1

Chymotrypsin

Answer 1

• ester
• digestive enzyme secreted by the pancreas
• serine protease
• carboxyl-terminal side of an aromatic or large hydrophobic residue

Question 2

2 catalytic strategies of chymotrypsin

Answer 2

• covalent catalysis

* general acid-base catalysis

Question 3

Proteases cleave proteins by

Answer 3

hydrolysis – addition of water to a peptide bond

Question 4

… inactivates chymotrypsin

Answer 4

di-isopropyl-fluorophosphate

Question 5

Residue in the active site of chymotrypsin

Answer 5

Serine 195

also Histidine 57 and Aspartate 102

Question 6

Covalent catalysis in chymotrypsin

Answer 6

nucleophile attacks carbonyl carbon of substrate

nucleophile covalently attached to substrate briefly

Question 7

Kinetics of chymotrypsin monitored

Answer 7

• acts on substrate analog that forms a colored product
• p-Bitrophenolate turns yellow, can be measured
(chromogenic substrate)

Question 8

Chymotrypsin in 2 phases

Answer 8

1. acylation to form acyl enzyme intermediate – quick
2. deacylation to regenerate free enzyme – slow
   • explained by formation of a covalently bound enzyme-substrate intermediate
   • use binding energy of substrate to accelerate the reaction

Question 9

Serine held in active site by

Answer 9

disulfide bonds

• in a cleft next to histidine and aspartate = more reactive

Question 10

Chymotrypsin shape

Answer 10

spherical

3 polypeptide chains linked with disulfide bonds

Question 11

Catalytic triad

Answer 11

• side chain of serine-195 hydrogen bonded to imidazole ring of histidine-57
• NH of this ring hydrogen bonded to carboxylate group of aspartate-102

Question 12

Alkoxide ion (O-)

Answer 12

• H polarizes serine’s hydroxyl group
• substrate -> histidine takes proton from serine
• aspartate makes histidine a better proton acceptor

Question 13

Stage 1 – acylation of the enzyme

Answer 13

1. substrate binding
2. nucleophilic attack of serine on the peptide carbonyl group
   • O on side chain of ser attacks carbonyl c
   • 4 atoms on C = tetrahedral intermediate
   • - charge on O = oxyanion hole – stabilized by interactions with NH groups from the protein
3. collapse of the tetrahedral intermediate to form the acyl-enzyme
   • transfer of the proton (H) from his to the amino group formed by cleavage of the peptide bond
4. release on the amine component
   • left with acyl-enzyme

Question 14

Stage 2 – deacylation of the ezyme

Answer 14

1. water binding
   • takes space from amine molecule
2. nucleophilic attack of water on the acyl-enzyme intermediate
   • His takes proton (H) from water
   • OH- attacks carbonyl C of acyl à tetrahedral intermediate
3. collapse of the tetrahedral intermediate
   • forms carboxylic acid product
4. release of the carboxylic acid intermediate
   • readies enzyme for another round of catalysis

Question 15

S1 (sub) pocket

Answer 15

deep hydrophic pocket of chymotrypsin into which the long, uncharged side chains of residues can fit
• chymotrypsin prefers bonds just past residues with large, hydrophobic side chains
* the binding of an appropriate side chain into this pocket positions the adjacent peptide bond into the active site for cleavage

Question 16

The specificity of chymotrypsin depends on

Answer 16

which amino acid is directly on the amino-terminal side of the peptide bond to be cleaved

Question 17

Bond to be cleaved

Answer 17

scissile bond

Question 18

Numbering to amino side

Answer 18

Residues to the amino terminal side of the sessile bond numbered
P1 P2 P3 (subs)
corresponding sites on enzyme numbered
S1 S2 S3 (subs)

Question 19

Numbering to carboxyl side

Answer 19

Residues to the carboxyl side of the sessile bond numbered
P1’ P2’ P3’ (subs)
corresponding sites on enzyme numbered
S1’ S2’ S3’

Question 20

Chymotrypsin cleaves at peptide bonds

Answer 20

after residues with an aromatic or nonpolar side chain

Question 21

Trypsin cleaves at peptide bonds

Answer 21

after residues with long positive side chains

eg Arginine, Lysine

Question 22

Elastase cleaves at peptide bonds

Answer 22

after amino acids with small side chains

eg Alanine, Serine

Question 23

Trypsin’s active site

Answer 23

• Aspartate 189 instead of serine

* attracts and stabilizes a positive arginine/lysine in substrate

Question 24

Elastases active site

Answer 24

* 2 residues at top replaced with bulkier valine residues (216, 190) * close to mouth of pocket = only small side chains enter

Question 25

Subtilisim

Answer 25

• protease in bacteria • catalytic triad and oxyanionic hole BUT 1 of NH that forms oxyanion hole forms side chain of aspartate instead of polypeptide backbone (convergent evolution)

Question 26

In addition to serine, 3 approaches to peptide bond hydrolysis

Answer 26

strategy – to generate a nucleophile that attacks the peptide carbonyl group • cysteine proteases • aspartyl proteases • metalloproteases

Question 27

Cysteine proteases

Answer 27

* Histidine activates cystein- acts as nucleophile that attacks the peptide bond * sulphur better nucleophle than oxygen is in serine * only need His and Cys = not triad * eg papain

Question 28

Aspartyl proteases

Answer 28

• 2 aspartic cid residues allow water to attack peptide bond 1st (deprotonated) – activates water by posing it for deprotonation 2nd (protonated) – polarizes peptide carbonyl = more susceptible to attack • may have existed as 2 separate units (2 copies of gene for ancestral enzyme • eg HIV and retroviruses

Question 29

Metalloproteases

Answer 29

* active site with bound metal ion (esp Zinc) * activates water to act as a nucleophile to attack the peptide carbonyl group * eg carboxypeptidase A

Question 30

Cysteine, Aspartyl, and Metalloproteases cleave peptide bonds

Answer 30

active site with features to 1. activate a water molecule or other nucleophile 2. polarize the peptide carbonyl group 3. stabilize a tetrahedral intermediate

Question 31

HIV protease

Answer 31

* protease inhibitor * dimeric aspartyl protease * cleaves multidomain viral proteins into their active forms

Question 32

Indinavir

Answer 32

* HIV protease inhibitor * blocks HIV protease from cleaving into functional units = virus not infectious * resembles peptide substrate of HIV protease * alcohol mimics tetrahedral intermediate * enters active site, adopts conformation that approximates symmetry of the enzyme * OH of alcohol interacts with 2 aspartate residues of active site

Question 33

Protease inhibitors used as drugs

Answer 33

must be specific for 1 enzyme without inhibiting other proteins in the body to prevent side effects