Chapter 7.3 Chymotrypsin Flashcards
list the 3 important structures of chymotrypsin
- hydrophobic substrate specificity pocket
- oxyanion hole
- active site
what does the hydrophobic substrate specificity pocket of chymotrypsin do?
recognizes aromatic amino acid residues (Phe, Tyr, Trp)
describe the oxyanion hole of chymotrypsin
two hydrogen bonds stabilize the tetrahedral intermediate oxyanion
what 2 hydrogen bonds stabilize the tetrahedral intermediate oxyanion hole of chymotrypsin?
backbone NH of Gly 193 and Ser 195
what makes up the active site of chymotrypsin?
the catalytic triad
what does the active site of chymotrypsin do?
cuts the peptide bond after aromatic amino acids in C terminal cleavage after backbone C=O
what do serine proteases do?
cut peptide bonds using serine
where is the active site of serine proteases? why?
on the surface of the protein for easy activity
what is a catalytic triad?
the three residues in an active site; the main residues that catalyze a reaction
what types of catalysis do serine proteases use? (2)
- acid base catalysis
- covalent catalysis
give an example of a serine protease
chymotrypsin
how is the catalytic triad of chymotrypsin held together?
by a hydrogen bond network
what 3 residues are in the catalytic triad of chymotrypsin?
- Asp102
- His57
- Ser195
what is the role of Asp102 in the catalytic triad of chymotrypsin? (2)
- makes His a better base
- maintains negatively charged protonation state
how does Asp102 make His57 a better base?
via a hydrogen bond with the nonprotonatable NH of His57
does Asp102 change form during the reaction of chymotrypsin?
nope; remains deprotonated throughout
what is the role of His57 in the catalytic triad of chymotrypsin?
act as a general acid (in protonated form) and base (in deprotonated form)
what is the role of Ser195 in the catalytic triad of chymotrypsin? (2)
- covalently attaches to substrate
- performs the cut
how many steps are in the chymotrypsin peptide cleavage mechanism?
6
what happens to the enzyme in the chymotrypsin peptide cleavage mechanism?
is fully regenerated at the end
what kind of specificity does the enzyme have in the chymotrypsin peptide cleavage mechanism? (2)
- geometric
- chemical
describe step 1 of the chymotrypsin mechanism where the polypeptide binds (AND GO DRAW IT)
polypeptide substrate binds to enzyme active site where the aromatic R group of the peptide binds to the substrate specificity pocket and Asp102 H-bonds to His57
what are the results of Asp102 H-bonding to His57 is step 1 of the chymotrypsin mechanism? (3)
- positions His57 correctly
- shifts electron density
- makes His57 a better base
describe step 2 of the chymotrypsin mechanism of the Ser195 nucleophilic attack (AND GO DRAW IT)
- His57 removes a proton from Ser195, which allows for a nucleophilic attack on the serine oxygen on the carbonyl carbon of the peptide
- His57 acts as a general base
- alkoxide is formed, which is a better nucleophile than alcohol
describe step 3 of the chymotrypsin mechanism where the C terminal fragment is released (3) AND GO DRAW IT
- His57 donates a proton to the amino group of the substrate, resulting in peptide bond cleavage
- the carboxyl-terminal fragment is released as the FIRST PRODUCT
- the FIRST TETRAHEDRAL INTERMEDIATE is formed as the oxyanion that is stabilized in the oxyanion hole made by backbone amino groups of Gly193 and Ser195
describe step 4 of the chymotrypsin mechanism of water’s (OH-) nucleophilic attack (3) AND GO DRAW IT
- water enters the active site
- His57 acts as a general base and removes a proton from water
- the resulting OH- acts as a nucleophile and attacks the carbonyl carbon of the covalent acyl-enzyme intermediate
describe step 5 of the chymotrypsin mechanism, release of the N-term fragment (3) AND DRAW IT
- His57 donates a proton (acting as a general acid) to Ser195, resulting in cleavage of the acyl-enzyme intermediate
- the amino-terminal fragment is released as the SECOND PRODUCT and the 2ND TETRAHEDRAL INTERMED is stabilized in the oxyanion hole made by backbone amino groups Gly193 and Ser195
- the catalytic triad is regenerated
describe step 6 of the chymotrypsin mechanism, or regeneration of the catalytic triad
the functional catalytic triad is regenerated within the enzyme active site
how are steps 1 and 6 of the chymotrypsin mechanism the same? (2)
- pocket and oxyanion hole empty
- catalytic triad H-bonded
how are steps 2 and 4 of the chymotrypsin mechanism the same? (3)
- His57 is a general base that takes a proton from whatever it is bonded to
- nucleophilic atatck of carbonyl carbon
- tetrahedral intermediate formed as product
how are steps 3 and 5 of the chymotrypsin mechanism the same? (4)
- His57 acts as a general acid and donates a proton to whatever it is bonded to
- bond cleavage on C-terminal side of carbonyl carbon
- tetrahedral intermediate broken down
- fragment leaves pocket
what are the 3 kinds of serine proteases?
- chymotrypsin
- trypsin
- elastase
describe the substrate binding pocket of chymotrypsin and what it accomodates
large substrate binding pocket accomodates larger aromatic residues (Tyr, Trp, Phe)
describe the substrate binding pocket of trypsin and what it accomodates
binding pocket has a negatively charged residue at the bottom which accomodates residues with a positive charge (Arg, Lys)
describe the substrate binding pocket of elastase and what it accomodates
contains threonine and valine residues that close off the binding pocket so that only small residues such as alanine can be accomodated
