Kinetic studies of the acylation of pig muscle D-glyceraldehyde 3-phosphate dehydrogenase by 1,3-diphosphoglycerate and of proton uptake and release in the overall enzyme mechanism.

Abstract

In the presence of NAD(+) the acylation by 1,3-diphosphoglycerate of the four active sites of pig muscle d-glyceraldehyde 3-phosphate dehydrogenase can be monitored at 365nm by the disappearance of the absorption band present in the binary complex of NAD(+) and the enzyme. A non-specific salt effect decreased the acylation rate 25-fold when the ionic strength was increased from 0.10 to 1.0. This caused acylation to be the rate-limiting process in the enzyme-catalysed reductive dephosphorylation of 1,3-diphosphoglycerate at high ionic strength at pH8. The salt effect permitted investigation of the acylation over a wide range of conditions. Variation of pH from 5.4 to 8.6 produced at most a two-fold change in the acylation rate. One proton was taken up per site acylated at pH8.0. By using a chromophoric H(+) indicator the rate of proton uptake could be monitored during the acylation and was also almost invariant in the pH range 5.5-8.5. Transient kinetic studies of the overall enzyme-catalysed reaction indicated that acylation was the process involving proton uptake at pH8.0. The enzyme mechanism is discussed in the light of these results.