BIOC 095 The Importance Of A Structural Hydrogen-bond To Catalysis In Thioredoxin Reductase

Question 1

Leonid Povolotskiy & Robert Hondal

Answer 1

Date: 20 Oct, 2017
Time: 2:30pm-

Question 2

Thioredoxin (Trx)

Answer 2

• Small enzyme found in nearly all known organisms
• Used as an antioxidant
• DNA synthesis = Possible cancer targets

Question 3

Thioredoxin reductase (TR)

Answer 3

• 2 Major subtypes
  
  1. High molecular weight
  2. Low molecular weight
• The mammalian form contains a selenocysteine residue as penultimate amino acid

Question 4

Why selenium?

Answer 4

• Betting at giving up and accepting e- than sulfur

- Resists permanent oxidative deactivation

Question 5

Sulfur TRs

Answer 5

• Exhibit almost the same catalytic properties
• How do sulfur containing TRs manage to do this?
• Hypothesis: C-terminal disulfide is strained due to the beta-turn beta motif

Question 6

Experimental design

Answer 6

• Replace N in wild type with O
• Use N-term from recombinant CeTR
  > Truncated by the final 8 amino acids
• Synthesize wild type C-term peptide and mutable C-term peptide

Question 7

Selective intramolecular disulfide formation

Answer 7

• Use novel cysteine protecting group on the sulfur

> Fmoc-Cys(STmp)

Question 8

Floc-Cys(STmp) advantages

Answer 8

• Can selectively the protect cysteine residues while leaving other residues intact

Question 9

Mutant peptide hydrolysis

Answer 9

• See if intramolecular disulfide could be made via air oxidation
• Check the stability at pH6, 7 and 8 in ammonium bicarbonate
  (0. 1M, pH 6 - T1, 7 - T2, 8 - T3)
• Increase pH > increase truncated products

Question 10

Conclusions

Answer 10

• NOT perform an air oxidation
• Ester bond = very label
• Semi synthesis approach scrapped