Crystalline d-glyceraldehyde 3-phosphate dehydrogenase from lobster tail contains 4 moles of NAD+ bound and reacts specifically with 4 moles of iodoacetic acid/mole of tetramer. The essential thiol group of d-glyceraldehyde 3-phosphate dehydrogenase appears to react with iodoacetic acid with a rate constant for the overall process that is independent of the extent of carboxymethylation. The d-glyceraldehyde 3-phosphate dehydrogenase–NAD+ absorption band has a variable molar extinction coefficient in the presence of phosphate that may be correlated with a proton dissociation of pK 6·86. The binding of NAD+ to d-glyceraldehyde 3-phosphate dehydrogenase weakens as alkylating agents react with the enzyme, and NAD+ promotes the reactivity of the essential thiol group. It is suggested that, on binding to d-glyceraldehyde 3-phosphate dehydrogenase, NAD+ lowers the pK of the essential thiol group, resulting in a catalytic role of NAD+ in the reaction catalysed by d-glyceraldehyde 3-phosphate dehydrogenase. If this theory is correct, then it is likely that a proton will be liberated during the phosphorolysis of the acyl-enzyme rather than in the redox step.
