On the occurrence, equilibria, and site of acyl-enzyme formation of glyceraldehyde-3-phosphate dehydrogenase.

Researches in several laboratories have given evidence that catalysis of aldehyde oxidation by glyceraldehyde-3-phosphate (G-3-P) dehydrogenase involves formation of an acyl-enzyme intermediate which is subsequently cleaved by inorganic orthophosphate (Pi) to give an acyl phosphate (l-5). Recently Warburg et al. (6) have expressed doubt that present evidence warrants acceptance of the acyl-enzyme theory in preference to the earlier suggestion (7, 8) that Pi was added to 3-phosphoglyceraldehyde to give an intermediate which was subsequently dehydrogenated to form an acyl phosphate. Their principal objection was based on measurements of the extent of diphosphopyridine nucleotide (DPN+) reduction immediately following the mixing of glyceraldehyde and the dehydrogenase from yeast. The extent of reduction in the average time required for an enzyme molecule to “turnover” once in the complete reaction system was always greater in the presence than in the absence of Pi. Experimental and theoretical data which reconcile this result with the acyl-enzyme theory are presented in this paper. A second phase of the research presented herein concerns further evidence that the acyl group is probably attached to a sulfhydryl group of the enzyme. The necessity of intact sulfhydryl groups for catalytic action, the demonstrations that substrate can protect the enzyme against inactivation by iodoacetate (IAA) (2,4), and the isolation of acetylglutathione from “acyl-enzyme” preparations (Y), together with the known properties of acyl coenzyme A and other acyl-S derivatives, suggest that the acyl group is attached to the enzyme as a thiol ester. However, alternative possible sites of attachment need to be considered; for example, Stadtman (10) has pointed out that acyl-imidazole derivatives may show relatively high values for the AF of hydrolysis. Other results prcscnted provide additional evidence that the acyl enzyme