GH mechanisms

Question 1

How do GHs generate H+ and OH- for catalysis?

Answer 1

Thorugh the use of 2 amino acids

Question 2

What are the 2 amino acids used in acid-base assisted catalysis?

Answer 2

Glutamate

Aspartate

Question 3

What does the single displacement mechanism do to the anomeric configuration of the glycone sugar?

Answer 3

Inverts it

Question 4

Describe the acid catalyst in the single displacement mechanism

Answer 4

Uncharged

The H is in the perfect position to be donated to the glycosidic oxygen

Question 5

Describe the catalytic base in the single displacement mechanism

Answer 5

Extracts H+ from water

OH- is in the perfect position to attack the C1 of the transition state

Question 6

How does the OH- attack in the single displacement mechanism?

Answer 6

Attacks from the opposite orientation to the glycosidic bond

Question 7

What is the distance between the catalytic base and catalytic acid?

Answer 7

10.5Å

Question 8

Why is the catalytic acid uncharged at neutral pH in the single displacement mechanism?

Answer 8

The amino acid environment is non-polar

Question 9

Why is the catalytic base charged at pH in the single displacement mechanism?

Answer 9

The carboxylic acid side chain is charged in a normal polar environment

Question 10

What does the double displacement mechanism do to the anomeric configuration of the glycone sugar?

Answer 10

Retention of the anomeric configuration of the glycone sugar

Question 11

What are the 2 distinct reactions in the double displacement mechanism?

Answer 11

Glycosylation

Deglycosylation

Question 12

Describe glycosylation

Answer 12

The catalytic acid-base is uncharged and the H is donated to the glycosidic oxygen
The C1 is attacked by the charged catalytic nucleophile
The glycosidic bond is cleaved
The aglycone is released from the active site and the catalytic acid-base is charged
The nucleophile is covalently attached to the glycone sugar at C1 (ester bond)
This is the glycosyl-enzyme intermediate

Question 13

Describe deglycosylation

Answer 13

The charged catalytic acid-base picks up H+ from water
The OH- generated attacks the ester bond and hydrolysed
The glycone sugar is released from the active site
The nucleophile is regenerated

Question 14

What is the distance between the catalytic amino acids in the double displacement mechanism?

Answer 14

5.5Å