CONICET | Buscador de Institutos y Recursos Humanos

NADP-dependent nonphosphorylating D-glyceraldehyde-3-phosphate dehydrogenase(EC 1.2.1.9) from spinach leaves has been purified to apparent electrophoretic homogeneityby ammonium sulfate fractionation, molecular sieving on Sephadex G-200,DEAE-cellulose, and 2,5-ADP-Sepharose affinity chromatography. The purified enzymeexhibited a specific activity of 15 pmol (mg protein)) min- and was characterizedas a homotetramer with a native molecular weight of 195,000. Preincubation of thepurified enzyme with NADP+ resulted in an almost twofold increase in enzymaticactivity. The rate of activation was slower than the rate of catalysis, indicating that theenzyme has hysteretic properties. This behavior results in a lag phase during activitymeasurement of the enzyme preincubated without NADP+. Substrate interaction andproduct inhibition studies suggest a rapid equilibrium random BiBi mechanism for thereaction. Thiol modifying reagents, iodoacetamide and diamide, completely inactivatedthe purified enzyme. Inactivation by iodoacetamide exhibited pseudo-first-order kineticswith a rate constant of 0.17 min-. D-Glyceraldehyde 3-phosphate effectivelyprotected the enzyme against inactivation by thiol reagents, suggesting that modificationoccurred at or near the substrate-binding site. Complete inactivation of the dehydrogenasewas correlated with incorporation of 8 mol [l-4C]iodoacetamide/mol enzyme.Total protection afforded by D-glyceraldehyde 3-phosphate against enzyme inactivationby iodoacetamide was correlated with a protection of 4 mol reactive residues/molenzyme. On the basis of these results it is suggested that one sulfhydryl group perenzyme subunit is essential for catalysis in spinach leaf nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase. A kinetic and molecular mechanism for the reactionis proposed. 0 1988 Academic Press, Inc.

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