Kinetic studies of oxidized nicotinamide–adenine dinucleotide-facilitated reactions of d-glyceraldehyde 3-phosphate dehydrogenase

[摘要]

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°C the rate of acylation of the NAD⁺-bound (or holo-) enzyme was 3.3×10⁵m⁻¹·s⁻¹ and the rate of phosphorolysis, the reverse reaction, was 7.5×10³m⁻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°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. PO₄³⁻ 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.