The 2'-phosphate of NADP is responsible for proper orientation of the nicotinamide ring in the oxidative decarboxylation reaction catalyzed by sheep liver 6-phosphogluconate dehydrogenase.
Sheep liver 6-phosphogluconate dehydrogenase shows a high specificity for NADP, with a much lower affinity for NAD. Discrimination between NADP and NAD suggests that the interactions between the 2'-phosphate and 6-phosphogluconate dehydrogenase contribute most of the binding energy for NADP. There are three active site residues, Asn-32, Arg-33, and Thr-34, that hydrogen-bond to the 2'-phosphate of NADP according to the crystal structure of the E.Nbr(8)ADP complex. In this study alanine mutagenesis was used to probe the contribution of each of the three residues to binding the cofactor and to catalysis. All mutant enzymes exhibit no significant change in V/E(t) or K(6PG) but an increase in K(NADP) that ranges from 6- to 80-fold. All mutant enzymes also exhibit at least a 7-fold increase in the primary kinetic (13)C-isotope effect-1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Asn-32, Arg-33, and Thr-34 in providing binding energy for NADP, and more importantly, the 2'-phosphate of NADP is required for proper orientation of the cofactor to allow rotation about the N-glycosidic bond as it is reduced in the hydride transfer step.
Li
Lei
L
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.
Cook
Paul F
PF
D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D006027:Glycosides / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D008099:Liver / Q000378:metabolism*; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry; D009249:NADP / Q000737:chemistry*; D009536:Niacinamide / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D010734:Phosphogluconate Dehydrogenase / Q000737:chemistry*; D012756:Sheep; D013912:Threonine / Q000737:chemistry
D006027:Glycosides; D010710:Phosphates; D009243:NAD; D009536:Niacinamide; D013912:Threonine; D009249:NADP; D001216:Asparagine; D010734:Phosphogluconate Dehydrogenase; D010100:Oxygen