Chapter 7.3 Chymotrypsin

Question 1

list the 3 important structures of chymotrypsin

Answer 1

1. hydrophobic substrate specificity pocket
2. oxyanion hole
3. active site

Question 2

what does the hydrophobic substrate specificity pocket of chymotrypsin do?

Answer 2

recognizes aromatic amino acid residues (Phe, Tyr, Trp)

Question 3

describe the oxyanion hole of chymotrypsin

Answer 3

two hydrogen bonds stabilize the tetrahedral intermediate oxyanion

Question 4

what 2 hydrogen bonds stabilize the tetrahedral intermediate oxyanion hole of chymotrypsin?

Answer 4

backbone NH of Gly 193 and Ser 195

Question 5

what makes up the active site of chymotrypsin?

Answer 5

the catalytic triad

Question 6

what does the active site of chymotrypsin do?

Answer 6

cuts the peptide bond after aromatic amino acids in C terminal cleavage after backbone C=O

Question 7

what do serine proteases do?

Answer 7

cut peptide bonds using serine

Question 8

where is the active site of serine proteases? why?

Answer 8

on the surface of the protein for easy activity

Question 9

what is a catalytic triad?

Answer 9

the three residues in an active site; the main residues that catalyze a reaction

Question 10

what types of catalysis do serine proteases use? (2)

Answer 10

1. acid base catalysis
2. covalent catalysis

Question 11

give an example of a serine protease

Answer 11

chymotrypsin

Question 12

how is the catalytic triad of chymotrypsin held together?

Answer 12

by a hydrogen bond network

Question 13

what 3 residues are in the catalytic triad of chymotrypsin?

Answer 13

1. Asp102
2. His57
3. Ser195

Question 14

what is the role of Asp102 in the catalytic triad of chymotrypsin? (2)

Answer 14

1. makes His a better base
2. maintains negatively charged protonation state

Question 15

how does Asp102 make His57 a better base?

Answer 15

via a hydrogen bond with the nonprotonatable NH of His57

Question 16

does Asp102 change form during the reaction of chymotrypsin?

Answer 16

nope; remains deprotonated throughout

Question 17

what is the role of His57 in the catalytic triad of chymotrypsin?

Answer 17

act as a general acid (in protonated form) and base (in deprotonated form)

Question 18

what is the role of Ser195 in the catalytic triad of chymotrypsin? (2)

Answer 18

1. covalently attaches to substrate
2. performs the cut

Question 19

how many steps are in the chymotrypsin peptide cleavage mechanism?

Answer 19

6

Question 20

what happens to the enzyme in the chymotrypsin peptide cleavage mechanism?

Answer 20

is fully regenerated at the end

Question 21

what kind of specificity does the enzyme have in the chymotrypsin peptide cleavage mechanism? (2)

Answer 21

1. geometric
2. chemical

Question 22

describe step 1 of the chymotrypsin mechanism where the polypeptide binds (AND GO DRAW IT)

Answer 22

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

Question 23

what are the results of Asp102 H-bonding to His57 is step 1 of the chymotrypsin mechanism? (3)

Answer 23

1. positions His57 correctly
2. shifts electron density
3. makes His57 a better base

Question 24

describe step 2 of the chymotrypsin mechanism of the Ser195 nucleophilic attack (AND GO DRAW IT)

Answer 24

1. His57 removes a proton from Ser195, which allows for a nucleophilic attack on the serine oxygen on the carbonyl carbon of the peptide
2. His57 acts as a general base
3. alkoxide is formed, which is a better nucleophile than alcohol

Question 25

describe step 3 of the chymotrypsin mechanism where the C terminal fragment is released (3) AND GO DRAW IT

Answer 25

1. His57 donates a proton to the amino group of the substrate, resulting in peptide bond cleavage 2. the carboxyl-terminal fragment is released as the FIRST PRODUCT 3. 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

Question 26

describe step 4 of the chymotrypsin mechanism of water's (OH-) nucleophilic attack (3) AND GO DRAW IT

Answer 26

1. water enters the active site 2. His57 acts as a general base and removes a proton from water 3. the resulting OH- acts as a nucleophile and attacks the carbonyl carbon of the covalent acyl-enzyme intermediate

Question 27

describe step 5 of the chymotrypsin mechanism, release of the N-term fragment (3) AND DRAW IT

Answer 27

1. His57 donates a proton (acting as a general acid) to Ser195, resulting in cleavage of the acyl-enzyme intermediate 2. 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 3. the catalytic triad is regenerated

Question 28

describe step 6 of the chymotrypsin mechanism, or regeneration of the catalytic triad

Answer 28

the functional catalytic triad is regenerated within the enzyme active site

Question 29

how are steps 1 and 6 of the chymotrypsin mechanism the same? (2)

Answer 29

1. pocket and oxyanion hole empty 2. catalytic triad H-bonded

Question 30

how are steps 2 and 4 of the chymotrypsin mechanism the same? (3)

Answer 30

1. His57 is a general base that takes a proton from whatever it is bonded to 2. nucleophilic atatck of carbonyl carbon 3. tetrahedral intermediate formed as product

Question 31

how are steps 3 and 5 of the chymotrypsin mechanism the same? (4)

Answer 31

1. His57 acts as a general acid and donates a proton to whatever it is bonded to 2. bond cleavage on C-terminal side of carbonyl carbon 3. tetrahedral intermediate broken down 4. fragment leaves pocket

Question 32

what are the 3 kinds of serine proteases?

Answer 32

1. chymotrypsin 2. trypsin 3. elastase

Question 33

describe the substrate binding pocket of chymotrypsin and what it accomodates

Answer 33

large substrate binding pocket accomodates larger aromatic residues (Tyr, Trp, Phe)

Question 34

describe the substrate binding pocket of trypsin and what it accomodates

Answer 34

binding pocket has a negatively charged residue at the bottom which accomodates residues with a positive charge (Arg, Lys)

Question 35

describe the substrate binding pocket of elastase and what it accomodates

Answer 35

contains threonine and valine residues that close off the binding pocket so that only small residues such as alanine can be accomodated