Role of nicotinamide adenine dinucleotide as an effector in formation and reactions of acylglyceraldehyde-3-phosphate dehydrogenase.

The equilibrium spectral and reactivity properties of a chromophoric acylglyceraldehyde-3-phosphate dehydrogenase (FA-GPDH) have been previously reported. Transient studies of these properties are reported herein. As with true-3-phosphoglyceroyl-enzyme these properties depend on the presence of bound coenzyme (NAD+). The reactivity of the acyl-enzyme toward acceptors (phosphate and arsenate) parallels the extent of its NAD+-induced spectral change [Malhotra, O. P., & Bernhard, S. A. (1973) Proc. Natl. Acad. Sci U.S.A. 70, 2077-2081]. The transcient deacylation of FA-GPDH, preincubated with NAD+, is kinetically biphasic. The relative amplitudes of the fast vs. the slow phase depend on NAD+ concentration but are independent of the nature and concentration of the acyl acceptor. At saturating NAD+ and acceptor concentrations, kinetic biphasicity persists. Perturbation of the acyl-apoenzyme spectrum by NAD+ is also kinetically biphasic. Evidence is presented that the NAD+-requiring acylation of the enzyme results in a protein conformation in which the acyl group is both spectrally perturbed and reactive toward acyl transfer. This acyl-enzyme undergoes a relatively slow isomerization to a conformation in which the acyl spectrum is unperturbed and unreactive in acyl transfer. These two acyl-enzyme conformations are also distinguished by their relative affinities for NAD+; hence, NAD+ is an effector of the conformational equilibrium. Kinetic biphasicity, wherever observed, can be accounted for in terms of two processes: (1) reactivity of the "active" acyl conformation and (2) slow isomerization of the inactive to the active conformation. The two acyl-enzyme conformers are present in finite albeit variable amounts dependent on the extent of NAD+ ligation. Evidence is presented suggesting that each of these conformers has a unique function.