Role of nicotinamide adenine dinucleotide as an effector in the formation and reactions of acyl-glyceraldehyde-3-phosphate dehydrogenase

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Abstract
The equilibrium spectral and reactivity properties of a chromophoric acylglyceraldehyde-3-phosphate dehydrogenase (FA-GPDH) were previously reported. Transient studies of these properties are reported here. As with true [sturgeon muscle] 3-phosphoglycerol-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. The transient 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. The NAD+-requiring acylation of the enzyme apparently 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 2 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 2 processes: reactivity of the active acyl conformation and slow isomerization of the inactive to the active conformation. The 2 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.