FIII: Chymsotrypsin: (Slides 1 - 20)

Question 1

What are ways that enzymes can increase reaction rates?

Answer 1

• Increasing the proximity - holding the reactants close together (Z increases) and the orientation (p)
• Using Chemical Catalysts
  Nucleophile Catalysts (lose electrons)
  Electrophile Catalysts (gain electrons)
  Acid Catalysts (Donate a proton)
  Base Catalysts (Gain a proton)
• Keep the transition state stable (lower activation energy)

Question 2

What is Chymotrypsin?

Answer 2

Chymotrypsin is a protease (enzyme) that has the ability to hydrolyze the peptide bonds of the aromatic amino acids: Phe, Tyr, Trp.

Question 3

Where does Chymotrypsin ‘weakly’ bind?

Answer 3

Chymotrypsin binds ‘weakly’ due to hydrogen bonds to the peptide chain (which acts as the substrate), upstream of the target amino acid

Question 4

What Terminus does chymotrypsin bind to?

Answer 4

Chymotrypsin will bind to the C-terminus (carboxylate group) of the Aromatic Amino Acids: Phenylalanine. Tyrosine and Tryptophan.

Question 5

When will Chymotrypsin not bind to these aromatic amino acids?

Answer 5

If any of these amino acids are followed by a proline.

Question 6

Where does strong binding of chymotrypsin occur?

Answer 6

Strong binding occurs in the binding pocket, when the benzene ring of these amino acids fit into the hydrophobic pocket, so the substrate (peptide chain) binds more tightly.

Question 7

What is the polarity of chymotrypsins binding pocket?

Answer 7

It is hydrophobic (non-polar)

Question 8

What does the binding pocket increase?

Answer 8

Proximity, increases Z as it binds the peptide chain more closely together (makes a good fit)

Question 9

What happens after the substrate (peptide chain) makes a good fit?

Answer 9

The peptide bond immediately following the target amino acid is going to be positioned right next to the catalytic unit

Question 10

What does the peptide chain following the target amino acid being placed right next to catalytic unit increase?

Answer 10

The orientation (increases P)

Question 11

What does H2O do?

Answer 11

H2O acts as a nucleophile (wants to give electrons) and attacks the electron deficient carbon, to donate its electrons.

Question 12

What happens to the Oxygen from the H20 that donated its electrons?

Answer 12

It becomes neutral (the oxygen is not a good nucleophile), making a unfavourable O+ transition state (becomes positive) and forming a bond with the C

Question 13

What does the Carbon that gets attacked by the nucleophile do?

Answer 13

It remains its stable 8 valence electrons, by giving the electrons to the upper O and withdrawing one of the bonds (no longer double bonded to upper O)

Question 14

How does the structure of the central carbon change?

Answer 14

It goes from sp2 (trigonal planar) to sp3 (tetrahedral)

Question 15

What is the transition state?

Answer 15

A semi-stable oxyanion (O negative upper bound C) and the carbon going from sp2 to sp3.

Question 16

How does the transition state breakdown?

Answer 16

The oxyanion (negative O) returns the bond to the carbon (reforms the double bond) and the carbon sends the electrons to the Nitrogen which forms the leaving group

Question 17

What happens to the Nitrogen leaving group?

Answer 17

It gains a proton from the surrounding environment and becomes NH2

Question 18

What is the second pathway that the transition state breakdown can take?

Answer 18

The oxyanion can return its electrons to the carbon, which then can return them to the carbon, then back to the original oxygen from the water causing the water molecule to break off (act as a leaving group) and no reaction occurs.

Question 19

What does peptide hydrolysis with H20 without a catalyst mean?

Answer 19

The enzyme chymotrypsin is not helping

Question 20

Why is hydrolysis by neural H2O not good?

Answer 20

• The H20 is not a good nucleophile as the oxygen is too electronegative
• The H20 makes a good leaving group, which can cause no reaction to occur.

Question 21

How slow is the hydrolysis with neutral H2O?

Answer 21

About one reaction every 10 years (excessively slow)

Question 22

What are the two easy steps for how Chymotrypsin can speed up the hydrolysis pf a peptide bond?

Answer 22

1. Chymotrypsin uses a nucleophilic group in the enzyme to attack the C = O bond.
   - Breaks the peptide bond releasing the C-terminal half of the group.
   - The N-terminal half remains covalently bonded to the enzyme group (acyl-enzyme intermediate)
2. Brings in H2O to release the N-terminal and restore the enzyme group to its original state.

Question 23

How many reactions per second can happen using chymotrypsin?

Answer 23

40 reactions per second.

Question 24

What is a Catalytic triad?

Answer 24

The catalytic triad is the three amino acids that line up side by side to produce a better nucleophile.

Question 25

What is the catalytic triad for Chymotrypsin?

Answer 25

Asp - 102
His - 57
Ser- 195

Question 26

What do the numbers indicate for the three amino acids in the catalytic triad?

Answer 26

The numbers indicate the position in the polypeptide sequence.

Question 27

Asp 102

Answer 27

A negative carboxylate, the negative charge on Asp 102 causes it to favour a positive charged partner, looking to become protonated.

Question 28

His 57

Answer 28

A weak base, can become positive if it captures a proton.

Question 29

Ser 195

Answer 29

Side Chain (OH) is not a good nucleophile, could give up a proton if it is shared with a lone pair with a suitable atom but can not do that by itself.

Question 30

What do these effects cause?

Answer 30

They cause Ser 195 to become a better nucleophile.

Question 31

In chymotrypsin what is the transition state stabilized from?

Answer 31

The oxyanion hole which consists of the backbone anion hole of Gly 193 and Ser 195

Question 32

What is Step 1 of the Catalytic triad?

Answer 32

The substrate binds to the enzyme with its target C=O group next to Ser 195
- His 157 acts as a general base, removing H+ from Ser 195
- Ser 195 becomes a better nucleophile and attacks the peptide C=O and binds with the carbon.
- Negative Charge on Asp 102 decolonizes the positive charge on His 57

Question 33

What does the oxyanion hole do?

Answer 33

The oxyanion hole (the N-H backbone of Gly 193 and Ser 195), pulls the O_ into the transition state (makes the oxygen have a negative charge)
- Complementary to the transition state (it is a requirement for catalysis)
- Changes bond angles to a tetrahedral configuration

Question 34

What is the breakdown of the first transition state?

Answer 34

Histidine now acts as a general acid, donating a proton to the N atom, so it can become a better leaving group.
- The ‘now’ NH group becomes the leaving group

Question 35

What happens with the formation of the Acyl-Enzyme intermediate?

Answer 35

The C-terminus is able to break off and leave

The N-terminal remains covalently bound to the acyl enzyme intermediate.

Question 36

What is Step 2 of the Catalytic Triad?

Answer 36

Water is able to now enter the catalytic site

His-57 acts as a general base, removing H+ from water

Water becomes a better nucleophile, attacks the acyl-enzyme C=O

Question 37

What is the formation of the second transition state?

Answer 37

The oxyanion hole stabilize the configuration state (makes the substrate oxygen negative (oxyanion))

Question 38

What is the Breakdown of the second transition state?

Answer 38

His 57 acts as a general acid donating H+ back to Ser 195

Able to break the acyl-enzyme bond.

Question 39

What is the final result?

Answer 39

The N-terminal half of the original peptide substrate now carries the target amino acid at its newly formed C-terminus.

Question 40

What is the formation of products?

Answer 40

Release the N-terminal half, and restore the enzyme group back to its original state.
N-terminal leaves
Catalytic triad is regenerated and ready to cycle again.

Question 41

What is the major role of His 57?

Answer 41

Acts as a general acid or base due to its pKa of 6.5 (close to physiological pH)

Question 42

What happens if you replace Ser with Ala?

Answer 42

Little catalytic reaction.

Question 43

How many times can this cycle repeat?

Answer 43

40 times per second.