Kinetic studies on oxidized nicotinamide--adenine dinucleotide-facilitated reactions of D-glyceraldehyde 3-phosphate dehydrogenase.

Abstract

The kinetics of the acylation of d-glyceraldehyde 3-phosphate dehydrogenase from pig muscle by 1,3-diphosphoglycerate in the presence of NAD(+) has been analysed by using the relaxation temperature-jump method. At pH7.2 and 8 degrees C the rate of acylation of the NAD(+)-bound (or holo-) enzyme was 3.3x10(5)m(-1).s(-1) and the rate of phosphorolysis, the reverse reaction, was 7.5x10(3)m(-1).s(-1). After a temperature-jump perturbation the equilibrium of NAD(+) binding to the acyl-enzyme was re-established more rapidly than that of the acylation. The rate of phosphorolysis of the apoacylenzyme from sturgeon muscle and of aldehyde release from the d-glyceraldehyde 3-phosphate-apoenzyme complex were </=40m(-1).s(-1) and </=12s(-1) respectively at pH8.0 and 22 degrees C, which means that both processes are too slow to contribute significantly to the reaction pathway of the reversible NAD(+)-linked oxidative phosphorylation of d-glyceraldehyde 3-phosphate. Phosphorolysis of both acyl-apoenzyme and acyl-holoenzyme was first-order in P(i) up to 100mm-P(i) and more. PO(4) (3-) could be the reactive species of the phosphorolysis of the acyl-holoenzyme, in which case phosphorolysis is a diffusion-controlled reaction, although other kinetically indistinguishable rate equations for the reaction are possible.