Importance in catalysis of the 6-phosphate-binding site of 6-phosphogluconate in sheep liver 6-phosphogluconate dehydrogenase.
The 6-phosphate of 6-phosphogluconate (6PG) is proposed to anchor the sugar phosphate in the active site and aid in orientating the substrate for catalysis. In order to test this hypothesis, alanine mutagenesis was used to probe the contribution of residues in the vicinity of the 6-phosphate to binding of 6PG and catalysis. The crystal structure of sheep liver 6-phosphogluconate dehydrogenase shows that Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 contribute a mixture of ionic and hydrogen bonding interactions to the 6-phosphate, and these interactions are likely to provide the majority of the binding energy for 6PG. All mutant enzymes, with the exception of T262A, exhibit an increase in K(6PG) that ranges from 5- to 800-fold. There is also a less pronounced increase in K(NADP), ranging from 3- to 15-fold, with the exception of T262A. The R287A and R446A mutant enzymes exhibit a dramatic decrease in V/E(t) (600- and 300-fold, respectively) as well as in V/K(6PG)E(t) (10(5) - and 10(4)-fold), and therefore no further characterization was carried out with these two mutant enzymes. No change in V/E(t) was observed for the Y191A mutant enzyme, whereas 20- and 3-fold decreases were obtained for the K260A and T262A mutant enzymes, respectively, resulting in a decrease in V/K(6PG)E(t) range from 3- to 120-fold. All mutant enzymes also exhibit at least an order of magnitude increase in 13C-isotope effect -1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 in providing the binding energy for 6PG. In addition, these residues also likely ensure proper orientation of 6PG for catalysis and aid in inducing the conformation change that precedes, and sets up the active site for, catalysis.
Li
Lei
L
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.
Dworkowski
Florian S N
FS
Cook
Paul F
PF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D005942:Gluconates / Q000737:chemistry*; D008099:Liver / Q000201:enzymology*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry*; D010734:Phosphogluconate Dehydrogenase / Q000737:chemistry*; D012756:Sheep
D005942:Gluconates; D010710:Phosphates; D010734:Phosphogluconate Dehydrogenase; C008884:6-phosphogluconic acid