Lab 7: Irreversible Inhibition

Question 1

What are the two proteolytic enzymes studied?

Answer 1

Trypsin and a-chymotrypsin

Question 2

What are the serine protease inhibitors?

Answer 2

PMSF, TPCK, and TLCK

Question 3

How do proteases work?

Answer 3

selectively hydrolyze the peptide bonds

Question 4

What makes the enzymes in the lab functional (be specific)?

Answer 4

• A catalytic triad made out of His57, Asp102 and Ser 195

- the secondary and tertiary protein structures bring the amino acids close together

Question 5

What does it mean when serine proteases can have endo- or exopeptidase activity? Give definitions

Answer 5

Endopeptidase: breaks peptide bonds in the protein molecule
Exopeptidase: breaks peptide bonds at the terminal ends of the protein molecule

Question 6

What determines the substrate specificity of trypsin and chymotrypsin?

Answer 6

A pocket adjacent to the catalytic triad

Question 7

What are the interactions formed with glycine for a-chymotrypsin?

Answer 7

The residue 215 and 226 (glycine’s) allow bulky side chains to extend inside the pocket
-a-chymotrypsin cavity lined by apolar amino acids that allows aromatic R groups (phenylalanine, tyrosine, tryptophan) to interact with the pocket via Van der Waals forces

Question 8

What does trypsin bind in the enzyme pocket?

Answer 8

The residue 215 and 226 (glycine’s) allow bulky side chains to extend inside the pocket
-tyrosine’s negative aspartic acid at position 189 allows for binding of positively charged lysine or arginine

Question 9

What does a-chymotrypsin cleave?

Answer 9

carboxyl side of phenylalanine, tyrosine, tryptophan except if followed by proline. also cleaves sometimes after large hydrophobic amino acid side chains

Question 10

what does trypsin cleave?

Answer 10

hydrolyzes peptide bonds on carboxyl side of lysine and arginine unless followed by proline

Question 11

What synthetic substrates are used in the lab?

Answer 11

BAPNA, GPPNA

Question 12

What is used to detect the enzyme cleaving the substrate?

Answer 12

-cleavage of one of the two peptide bonds yields p-nitro-aniline (yellow product detectable at A405nm)

Question 13

What is the type of irreversible inhibitor used in the lab and what does it stand for?

Answer 13

• use covalent irreversible serine protease inhibitors (serpins)

Question 14

What are the two steps of inhibition using serpins?

Answer 14

1) non-covalent enzyme-inhibitor complex forms (binding causes reversible E-I complex)
2) formation of covalent bond that blocks the catalytically active amino acid residues

Question 15

What is the correct substrate for trypsin? Why?

Answer 15

BAPNA (has a arginine)

Question 16

What was the correct substrate for a-chymotrypsin?

Answer 16

GPPNA (has phenylalanine)

Question 17

What are the correct inhibitors for trypsin?

Answer 17

TLCK and PMSF

Question 18

What are the correct inhibitors for a-chymotrypsin?

Answer 18

PMSF, TPCK

Question 19

What was the control for substrate specificity? Was it a positive or negative control?

Answer 19

• DMF was the control (what the substrates were dissolved in)
• it was a negative control (do not expect the enzyme to hydrolyze the peptide bonds because it is not the correct substrate

Question 20

What was the control for inhibitor specificity? Was it a positive or negative control?

Answer 20

The control for inhibitor specificity was 30% methanol in assay buffer (the solvent used for the inhibitors)
It is a negative control because it does not give the desired outcome of inhibition of the enzyme