Nicotinamide Adenine Dinucleotide-specific Glyceraldehyde 3-Phosphate Dehydrogenase from Pisum sativum

The assay for NAD+-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) was critically examined in order to obtain reliable kinetic data. Product inhibition by 1,3-diphosphoglyceric acid was relieved by the inclusion of ADP, magnesium chloride, and 3-phosphoglyceric acid kinase in the assay. Reactions were initiated with glyceraldehyde 3-phosphate since this substrate was found to be unstable under assay conditions. Rate measurements were made within 20 s of the addition of this substrate. Initial velocity and product inhibition studies indicated two possible Bi Uni Uni Uni Ping Pong kinetic mechanisms, and one of these (NAD+ on, phosphate on, 1,3-diphosphoglyceric acid off, glyceraldehyde 3-phosphate on, NADH off) was chosen on the basis of published studies of the enzyme which have indicated that a stable acyl enzyme is an obligatory intermediate in the reaction. The proposed mechanism predicts the existence of a stable enzyme-NAD+ complex, and this form is well known. The mechanism agrees with many of the published observations on the enzyme but differs from those indicated by previous kinetic studies. It is suggested that these workers have not fully considered the problems associated with the assay, and that this can account for these discrepancies.