IT - Enzymes II

Question 1

What are the 4 steps involved in TIM - acid base catalysis?

Answer 1

1) Substrate Binding: DHAP binds to the active site of TIM.

2) Proton Transfer:

• A basic amino acid residue (B1) in TIM abstracts a proton from the C1 hydroxyl group of DHAP.
• This forms an enediol intermediate.
• An acidic amino acid residue (A2) in TIM donates a proton to the C2 carbonyl oxygen of the enediol intermediate.

3) Proton Transfer: The reverse of step 2 occurs.

• B2 abstracts a proton from the C1 hydroxyl group of the enediol intermediate.
• A1 donates a proton to the C2 carbonyl oxygen of the enediol intermediate.

4) Product Release: G3P dissociates from TIM

Question 2

What is the role of Glu165 in TIM?

Answer 2

Glu165 acts as the catalytic base in TIM

Question 3

How important is Glu165 for TIM activity?

Answer 3

Mutations in Glu165 significantly reduce catalytic rate (Glu165Ala: 1 million times slower, Glu165Asp: 1 thousand times slower)

Question 4

How does TIM prevent unwanted side reactions?

Answer 4

TIM traps and stabilizes the enediol intermediate, preventing its conversion to glyoxal (undesirable product)

Question 5

How does the pink loop in TIM contribute to catalysis?

Answer 5

The pink loop occludes the active site from environment, increasing the catalytic rate by 100,000 fold

Question 6

What is the significance of the (ba)8-barrel fold?

Answer 6

It’s the most common enzymatic fold, found in 10% of enzymes, and a potential starting point for new enzyme functions

Question 7

How can the (ba)8-barrel be relevant for biotechnology?

Answer 7

Understanding this fold can help design catalysts for new reactions

Question 8

How does the (ba)8-barrel fold evolve? (2)

Answer 8

convergent evolution

• different functions converge into the same fold

divergent evolution

• common ancestor structure diverges into different function

Question 9

What suggests divergent and convergent evolution in (ba)8-barrels?

Answer 9

Divergent:

• Common structural features from a common ancestor

Convergent

• Diverse functions without significant sequence similarity

Question 10

Describe the biosynthesis of Histidine?

Answer 10

PPRP + ATP is converted to the ImGP / AICAR precursors

• ImGP is used in histidine biosynthesis (His)
• AICAR is used in purine biosynthesis (GTP, ATP)

Question 11

What is the role of HisA and HisF in histidine formation?

Answer 11

HisA catalyses the formation of a precursor that is converted by HisF to form ImGP and AICAR

• HisF is a branch-point enzyme!

Question 12

What hypothesis was put forward for the gene duplication for (ba)8 barrel? (2)

Answer 12

Hypothesis put forward:

• Gene duplication generates two initially identical half-barrels; the half barrels are fused and adapted into an ancestral (ba)8 barrel
• Gene duplication leads to diversification into two enzymes with distinct catalytic activities

Question 13

Can enzymatic activities be interconverted in the laboratory? (2)

Answer 13

• HisF show cross reactivity to catalyse the HisA reaction
• TrpF and HisA catalyze similar isomerization reactions