On the function of half-site reactivity: Intersubunit NAD +-dependent activation of acyl-glyceraldehyde 3-phosphate dehydrogenase reduction by NADH

Abstract

Glyceraldehyde 3-phosphate dehydrogenase extracted from sturgeon muscle, exhibits half-site reactivity in its reaction with the pseudo-substrate β -(2-furyl) acryloyl phosphate ( Malhotra & Bernhard, 1968 ). The product is the difurylacryloyl thiol ester enzyme tetramer formed from the active site cysteinyl residue. The electronic spectrum of the furylacryloyl thiol ester linkage is perturbed upon binding of oxidized coenzyme (NAD + ) at the acyl site ( Malhotra & Bernhard, 1973 ). Likewise, we now report the perturbation in electronic spectrum of this furylacryloyl thiol ester upon interaction with NADH at the acyl site. The spectral perturbation accompanying NADH binding is large and in the opposite direction from that due to the binding of NAD + . The “color” of the chromophoretic acyl-enzyme linkage is a reflection of its chemical properties: furylacryloyl-enzyme-NAD complex is “red-shifter” (λ max =360 nm), and reacts uniquely with inorganic phosphate to yield the corresponding acyl phosphate. Furylacryloyl-enzyme-NADH complex is “blue-shifter” (λ max =330 nm) and is uniquely capable of reduction by NADH. Despite the presence of a ubiquitous NADH-induced color change, NADH reduction occurs only when NAD is bound to the remaining two non-acylated subunits. Hence, these results provide a direct example of allosteric regulation of enzyme function by ligand interaction at an adjacent subunit site. This site is known to be at least 17 A removed from the site of reduction ( Buehner et al. , 1974 ). Carboxymethylation of the two remaining (active) thiol groups of the diacyl-enzyme tetramer, a process which modifies the affinity for NAD at this site to only a small extent, does not affect the NAD-dependent activation of the acyl-NADH site for reduction. Qualitative transient kinetic evidence suggests that NAD binding to the non-acylated sites favors a slow conformational change leading to reducibility at acyl sites. The affinity of the various sites, acylated and non-acylated, for coenzyme ligands varies with the extent of acylation and the extent and nature of the ligand occupancies at adjacent subunits. Since the molar concentration of glyceraldehyde 3-phosphate dehydrogenase sites in muscle sarcoplasmic fluid is high (of the order of 1 pm), these results suggest that the “enzyme” functions as a regulatory warehouse for energy via acyl-enzyme-ATP interconversion and NADH/NAD redox potential regulation.