Oxidative Decarboxylation of 6-Phosphogluconate by 6-Phosphogluconate Dehydrogenase Proceeds by A Stepwise Mechanism with NADP and APADP as Oxidants

Document Type

Article

Publication Date

9-1-1998

Publication Title

Biochemistry

Abstract

Primary kinetic deuterium, 13C, and multiple deuterium/13C-isotope effects on V/K6PG have been measured for the Candida utilis (cu) and sheep liver (sl) 6-phosphogluconate dehydrogenases (6PGDH). With NADP as the dinucleotide substrate, the following values of D(V/K6PG), 13(V/K6PG)H, and 13(V/K6PG)D were measured at pH 8 for cu6PGDH (sl6PGDH): 1.57 ± 0.08 (1.87 ± 0.10), 1.0209 ± 0.0005 (1.0059 ± 0.000 10), 1.0158 ± 0.0001 (1.0036 ± 0.0008). With APADP as the dinucleotide substrate, values for the above isotope effects at pH 8 are as follows: 2.98 ± 0.08 (2.47 ± 0.06), 1.0106 ± 0.0002 (1.0086 ± 0.000 09), and 0.9934 ± 0.0003 (0.9950 ± 0.0003). Results indicate the oxidative decarboxylation of 6PG to the 1,2-enediol of ribulose 5-phosphate proceeds via a stepwise mechanism with hydride transfer preceding decarboxylation in all cases. The inverse 13C-isotope effect observed with APADP and 6PG-3d may reflect a preequlibrium isotope effect on the binding of 6PG preceding hydride transfer. Deuterium-isotope effects on V, V/KNADP, and V/K6PG are identical at all pHs and for both enzymes. The primary deuterium-isotope effect on V/K6PG for both enzymes is constant at pH values below the pK in the pH profile for V/K6PG, and decreases as the pH increases. Data suggest the development of rate limitation by a step or steps other than the hydride-transfer step as the pH is increased.

Comments

This work was supported by grants from the National Institutes of Health to P.F.C. (GM 50872) and W.W.C. (GM 18938).

DOI

10.1021/bi980611s

Volume

37

Issue

36

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