Half-site reactivity in 6-phosphogluconate dehydrogenase from human erythrocytes.
6-Phosphogluconate dehydrogenase from human erythrocytes was purified by an improved procedure. Binding studies showed that the dimeric enzyme binds 2 mol of NADP+/mol but only 1 mol of NADPH/mol, and that the bindings of oxidized and reduced coenzyme are mutually exclusive. From initial-rate kinetics and inhibition studies, a sequential random-order mechanism is proposed. Double-reciprocal plots with NADP+ as varied substrate show a downward curvature, indicating a negative co-operativity. We suggest that the negative co-operativity observed kinetically is a result of the half-site reactivity for the NADPH. The different binding stoichiometries for NADP+ and NADPH generate a non-linear relationship between the apparent dissociation constant for the NADPH and the concentrations of the NADP+, resulting in a regulatory mechanism highly sensitive to the changes in the NADP+/NADPH ratio.
D001665:Binding Sites; D004912:Erythrocytes / Q000201:enzymology*; D005942:Gluconates / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010734:Phosphogluconate Dehydrogenase / Q000037:antagonists & inhibitors / Q000097:blood*
D005942:Gluconates; C045158:gluconate 6-sulfate; D009249:NADP; D010734:Phosphogluconate Dehydrogenase