All mutant enzymes in the 2'-phosphate site exhibit an increase in KNADP that ranges from 6-fold to 80-fold. A 7-fold increase in the primary kinetic 13C-isotope effect minus 1 in all mutant enzymes indicates that the decarboxylation step has become more rate-limiting. All mutant enzymes in the 6-phosphate site, with the exception of T262A exhibit an increase in K 6PG that ranges from 5-fold to 800-fold. The R287A and R446A mutant enzymes exhibit a dramatic decrease in V/Et (600-fold and 300-fold, respectively) as well as in V/K6PGEt (105-fold and 104-fold). All mutant enzymes also exhibit at least an order of magnitude increase in 13C-isotope effect minus 1, indicating that the decarboxylation step has become more rate-liming. The S128A, H186A and N187A mutant enzymes exhibit a decrease in V/Et, ranging from 7-fold to 67-fold. An increase in K6PC was observed for S128A and H187A mutant enzymes. An increased Ki NADPH was measured for all of the mutant enzymes, and all mutant enzymes also exhibit an increase in the primary kinetic 13C-isotope effect minus 1, indicating that the decarboxylation step has become more rate-limiting.

Data are consistent with significant roles for N32, R33 and T34 in providing binding energy for NADP, and Y191, K260, T262, R287 and R446 in providing the binding energy for 6PG. In addition, these residues also likely ensure proper orientation of NADP/6PG for catalysis and aid in inducing the conformation change that precedes catalysis. Data also suggest that residues S128, H186 and N187 help to guide the isomerization of the cofactor and provide binding energy for NADPH.

Sheep liver 6-phosphogluconate dehydrogenase (6PGDH) shows a high specificity for NADP. Discrimination between NADP and NAD suggests that the interactions between the 2'-phosphate and 6PGDH contribute most of the binding energy for NADP. 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. Crystal structure of the E:Nbr8ADP complex shows that Asn32, Arg33 and Thr34 are within hydrogen-bonding distance to the 2'-phosphate of NADP. And residues Y191, K260, T262, R287 and R446 are in the vicinity of the 6-phosphate of 6PG in the E:6PG binary complex. Residues S128 and N187 are within hydrogen-bonding distance to both 6PG in the E:6PG binary complex and NADPH in the E:NADPH binary complex, suggesting that these two residues may play a dual role during the catalytic reaction. In addition, residue H186 also hydrogen bonds to NADPH in the E:NADPH binary complex, while in the E:6PG binary complex it is within hydrogen-bonding distance to S128 and N187. In this study, alanine mutagenesis was used to probe the contribution of each of the eleven residues to binding the cofactor/substrate and to catalysis.