Question

A. GAPDH catalyzes the reversible conversion of glyceraldehyde 3-phosphate((O3)POC-CHOH-COH) to 1,3-bisphosphoglycerate ((O3)POC-CHOH-COP(O3)). Write the mechanism of the GAPDH reaction. Be sure to include any important amino acid side chains that are directly involved in the reaction and any cofactors.

Answer 1

1. Glyceraldehyde-3-phosphate (GAP) binds to the enzyme

2. Nucleophilic attack of sulfhydryl group (SH) of Cys166 on the aldehyde forms a thio-hemiacetal. His194 also helps in this reaction.

3. Oxidation of this thio-hemiacetal by NAD+ produces NADH and acyl-thioester intermediate.

4 & 5. Nucelophilic attack of Pi on the acyl-thioester intermediate releases high-energy compound 1,3-bisphosphoglycerate (BPG) and NADH. NAD+ replaces NADH to regenerate active enzyme.