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On the function of half site reactivity inter subunit nad dependent activation of acyl glyceraldehyde 3 phosphate dehydrogenase ec reduction by nadh

Journal of Molecular Biology 108(1): 123-138

On the function of half site reactivity inter subunit nad dependent activation of acyl glyceraldehyde 3 phosphate dehydrogenase ec reduction by nadh

Glyceraldehyde 3-phosphate dehydrogenase [EC] extracted from sturgeon muscle, exhibits half-site reactivity in its reaction with the pseudo-substrate .beta.-(2-furyl) acryloyl phosphate. 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 NAD+ at the acyl site. The electronic spectrum of this furylacryloyl thiol ester is perturbed 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 (.lambda.max = 360 nm) and reacts uniquely with Pi to yield the corresponding acyl phosphate. Furylacryloyl-enzyme-NADH complex is blue-shifter (.lambda.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 2 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 at least 17 .ANG. removed from the site of reduction. Carboxymethylation of the 2 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-acrylated, 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 mM), these results suggest that the enzyme functions as a regulatory warehouse for energy via acyl-enzyme-ATP interconversion and NADH/NAD redox potential regulation.

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Accession: 006027346

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