title: Oxidative decarboxylation of 6-phosphogluconate by 6-phosphogluconate dehydrogenase proceeds by a stepwise mechanism with NADP and APADP as oxidants.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, USA.
authors.forename: C C
authors.initials: CC
authors.lastname: Hwang
authors.forename: A J
authors.initials: AJ
authors.lastname: Berdis
authors.forename: W E
authors.initials: WE
authors.lastname: Karsten
authors.forename: W W
authors.initials: WW
authors.lastname: Cleland
authors.forename: P F
authors.initials: PF
authors.lastname: Cook
mesh_terms: D000818:Animals; D002175:Candida / Q000201:enzymology; D002247:Carbon Isotopes; D003653:Decarboxylation; D003903:Deuterium / Q000737:chemistry; D005942:Gluconates / Q000737:chemistry*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000031:analogs & derivatives* / Q000737:chemistry*; D016877:Oxidants / Q000737:chemistry*; D010084:Oxidation-Reduction; D010734:Phosphogluconate Dehydrogenase / Q000737:chemistry*; D012756:Sheep; D013379:Substrate Specificity
chemical_list: D002247:Carbon Isotopes; D005942:Gluconates; D016877:Oxidants; C048483:3-acetylpyridine-adenine dinucleotide phosphate; D009249:NADP; D003903:Deuterium; D010734:Phosphogluconate Dehydrogenase; C008884:6-phosphogluconic acid