BB12 – Chymotrypsin and proteases Flashcards
Common catalytic strategies
- covalent catalysis
- general acid-base catalysis
- metal-ion catalysis
- catalysis by approximation
Chymotrypsin
- ester
- digestive enzyme secreted by the pancreas
- serine protease
- carboxyl-terminal side of an aromatic or large hydrophobic residue
2 catalytic strategies of chymotrypsin
- covalent catalysis
* general acid-base catalysis
Proteases cleave proteins by
hydrolysis – addition of water to a peptide bond
… inactivates chymotrypsin
di-isopropyl-fluorophosphate
Residue in the active site of chymotrypsin
Serine 195
also Histidine 57 and Aspartate 102
Covalent catalysis in chymotrypsin
nucleophile attacks carbonyl carbon of substrate
nucleophile covalently attached to substrate briefly
Kinetics of chymotrypsin monitored
• acts on substrate analog that forms a colored product
• p-Bitrophenolate turns yellow, can be measured
(chromogenic substrate)
Chymotrypsin in 2 phases
- acylation to form acyl enzyme intermediate – quick
- 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
Serine held in active site by
disulfide bonds
• in a cleft next to histidine and aspartate = more reactive
Chymotrypsin shape
spherical
3 polypeptide chains linked with disulfide bonds
Catalytic triad
- 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
Alkoxide ion (O-)
- H polarizes serine’s hydroxyl group
- substrate -> histidine takes proton from serine
- aspartate makes histidine a better proton acceptor
Stage 1 – acylation of the enzyme
- substrate binding
- 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 - 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 - release on the amine component
• left with acyl-enzyme
Stage 2 – deacylation of the ezyme
- water binding
• takes space from amine molecule - nucleophilic attack of water on the acyl-enzyme intermediate
• His takes proton (H) from water
• OH- attacks carbonyl C of acyl à tetrahedral intermediate - collapse of the tetrahedral intermediate
• forms carboxylic acid product - release of the carboxylic acid intermediate
• readies enzyme for another round of catalysis
S1 (sub) pocket
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
The specificity of chymotrypsin depends on
which amino acid is directly on the amino-terminal side of the peptide bond to be cleaved
Bond to be cleaved
scissile bond
Numbering to amino side
Residues to the amino terminal side of the sessile bond numbered
P1 P2 P3 (subs)
corresponding sites on enzyme numbered
S1 S2 S3 (subs)
Numbering to carboxyl side
Residues to the carboxyl side of the sessile bond numbered
P1’ P2’ P3’ (subs)
corresponding sites on enzyme numbered
S1’ S2’ S3’
Chymotrypsin cleaves at peptide bonds
after residues with an aromatic or nonpolar side chain
Trypsin cleaves at peptide bonds
after residues with long positive side chains
eg Arginine, Lysine
Elastase cleaves at peptide bonds
after amino acids with small side chains
eg Alanine, Serine
Trypsin’s active site
- Aspartate 189 instead of serine
* attracts and stabilizes a positive arginine/lysine in substrate
Elastases active site
- 2 residues at top replaced with bulkier valine residues (216, 190)
- close to mouth of pocket = only small side chains enter
Subtilisim
• 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)
In addition to serine, 3 approaches to peptide bond hydrolysis
strategy – to generate a nucleophile that attacks the peptide carbonyl group
• cysteine proteases
• aspartyl proteases
• metalloproteases
Cysteine proteases
- 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
Aspartyl proteases
• 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
Metalloproteases
- active site with bound metal ion (esp Zinc)
- activates water to act as a nucleophile to attack the peptide carbonyl group
- eg carboxypeptidase A
Cysteine, Aspartyl, and Metalloproteases cleave peptide bonds
active site with features to
- activate a water molecule or other nucleophile
- polarize the peptide carbonyl group
- stabilize a tetrahedral intermediate
HIV protease
- protease inhibitor
- dimeric aspartyl protease
- cleaves multidomain viral proteins into their active forms
Indinavir
- 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
Protease inhibitors used as drugs
must be specific for 1 enzyme without inhibiting other proteins in the body to prevent side effects
