Lecture 8B

Question 1

How do Enzymes participate in acid-base catalysis

Answer 1

AA’s in the enzyme active site can donate a proton (acid catalysis) or accept a proton (base catalysis)

Question 2

What is chymotrypsin?

Answer 2

Chymotrypsin is a protease that hydrolyzes peptide bonds. Chymotrypsin is secreted by cells in the pancreas in an inactive form, and is proteolyzed to an active form in the intestines where it hydrolyzes (digests/cleaves) proteins to aid in digestion.

Question 3

Chymotrypsin uses what kind of catalysis?

Answer 3

Chymotrypsin uses “base catalysis”: its catalytic histidine (His57) can abstract a proton away from its catalytic serine (Ser195) and also from water, activating their oxygens to act as nucleophiles. (to “abstract” a proton is to deprotonate)

Chymotrypsin also uses “covalent catalysis”: the activated/deprotonated
catalytic serine (Ser195 alkoxide ion) is a strong nucleophile

Question 4

Protein groups can serve as covalent catalysts due to the presence of

Answer 4

Lone electron pairs

Question 5

How do these protein groups serve as covalent catalysts

Answer 5

In the active sites of some enzymes, these groups become deprotonated and act as nucleophiles. They attack electron-deficient atoms on substrates and form covalent intermediates (i.e., transient compounds). Chymotrypsin and other serine proteases use their activated serine alkoxide ion to perform a nucleophilic attack on the peptide substrate, forming a covalent intermediate as a first step in hydrolyzing the peptide bond.

Question 6

What are serine proteases

Answer 6

enzymes that hydrolyze peptide bonds using an active site serine

Question 7

Proteases function to what? The bond that is cleaved is called?

Answer 7

proteases hydrolyze (cleave) peptide bonds - the bond that is cleave is the “scissile bond”

Question 8

serine proteases contain a “_______” that includes a catalytically active serine

Answer 8

“catalytic triad”

Question 9

Examples of serine proteases? Similarities and differences?

Answer 9

chymotrypsin, trypsin, elastase, subtilisin, carboxypeptidase A

• all use a similar enzyme mechanism
• have different substrate specificities (i.e., bind to different peptides within a protein so they cleave at different amino acid residues)
• many are related in amino acid sequence and structure (e.g., chymotrypsin, trypsin, elastase)
• not all serine proteases are related in sequence and structure (e.g., chymotrypsin and subtilisin) – are related by “convergent evolution” – different proteins that evolved similar mechanisms

Question 10

How and where do serine proteases cleave the peptide bond

Answer 10

Serine proteases are specific for the side chain of the amino acid on the N-terminal side of the peptide bond that will be cleaved (i.e., R1 on the blue segment, P2). They specifically recognize this side chain and cleave the peptide bond after this amino acid. The peptide N-terminal to the cleaved bond is the “P2” product as it is released from the enzyme second; the “P1” product is C-terminal to the cleavage site and it is released first

Question 11

Describe the process of chymotrypsin cleaving the peptide bond

Answer 11

Recognizes amino acids with bulky hydrophobic and/or aromatic side chains and cleaves the peptide bond that is C-terminal to these:

• Trp, Phe, Tyr (aka bulky aromatics)
• Met (aka bulky hydrophobic)

Question 12

Chymotrypsin binds bulky hydrophobic side chains via _____

Answer 12

its hydrophobic specificity pocket

Question 13

Hydrolysis takes place in how many stages

Answer 13

2

Question 14

1st stage of (a general) hydrolysis?

Answer 14

The enzyme binds to the peptide substrate and forms a “covalent intermediate” with one of the products, P2, and P1 is released - fast step (i.e., the enzyme forms a covalent bond with part of the peptide, designated P2, and the other part, P1, is released from the complex when the peptide bond is hydrolyzed)

Question 15

2nd step of (a general) hydrolysis

Answer 15

The second product, P2, is released from the enzyme - slow step

Question 16

Which is the slow step of hydrolysis?

Answer 16

2nd step

Question 17

In the 1st step of hydrolysis, the enzyme forms a covalent intermediate with which product?

Answer 17

P2 aka N-terminus

Question 18

The active site serine of chymotrypsin forms a covalent bond with the carbonyl carbon of the amide to form ____

Answer 18

an acyl-enzyme intermediate

Question 19

What is the 1st step of hydrolysis using chymotrypsin?

Answer 19

First the highly reactive Ser195 hydroxyl oxygen (alkoxide ion) in the chymotrypsin active site attacks the carbonyl carbon of the peptide substrate to form covalent bond with P2. The peptide bond is hydrolyzed, releasing P1, and leaving P2 covalently attached to the enzyme à acyl-enzyme intermediate.

Question 20

What is the 2nd step of hydrolysis using chymotrypsin?

Answer 20

Second, this new bond is hydrolyzed, releasing the carboxylic acid component of the substrate (P2) to regenerate the enzyme

Question 21

The hydrolysis reaction is thermodynamically ____?

Answer 21

Favourable

Question 22

The hydrolysis reaction proceeds quickly or slowly? Describe the activation energy of the 2 transition states

Answer 22

proceeds extremely slowly in the absence of enzyme. Chymotrypsin lowers the activation energy. Energy barriers (i.e., high-energy transition states), X‡ 1 and X‡ 2, exist at ES®E-P2 and E-P2®E+P2. The acyl-enzyme intermediate, E-P2, is a lower energy intermediate in the reaction

Question 23

What mates the active site serine particularly reactive?

Answer 23

• because of its position within a “catalytic triad”: Ser195, His57 and Asp102
• The catalytic triad activates Ser195 for chymotrypsin catalysis

Question 24

In the chymotrypsin active site, which part of the compounds are H bonded

Answer 24

the hydroxyl of Ser195 is H-bonded to His57 and the imidazole N-H of His57 is H-bonded to the carboxylate of Asp102

Question 25

His57 acts as a ____, abstracting a proton from Ser195-OH

Answer 25

“base catalyst”

Question 26

Ser195-O- can now act as a ___ after being deprotonated?

Answer 26

“covalent catalyst”

Question 27

Asp102 promotes catalysis by?

Answer 27

by stabilizing the resulting positively charged His57

Question 28

Example of divergent evolution and what is it?

Answer 28

Chymotrypsin, trypsin, elastase and thrombin (involved in the blood-clotting catalytic cascade) belong to the same family of serine proteases, having all descended from a common ancestor by gene duplication and mutation.

Question 29

Example of convergent evolution and what is it?

Answer 29

Subtilisin is not a sequence or structural homolog of the chymotrysin family but shares the catalytic triad and reaction mechanism. Still other proteases use the same mechanism but different nucleophile or base catalysis.