Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase.

The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.

Identification of an arginine residue in the dual coenzyme-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides that plays a key role in binding NADP+ but not NAD+.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides can utilize either NADP or NAD as coenzyme. The enzyme's three-dimensional structure has been solved (Rowland et al., 1994, Structure 2, 1073-1087) and shown to contain a conventional nucleotide binding domain. NADP+ was modeled into the structure by superimposing the beta alpha beta domain and that of coenzyme-bound 6-phosphogluconate dehydrogenase (Adams et al., 1994, Structure 2, 651-658), enabling us to identify Arg-46 as a potentially important residue for NADP+ binding. Using site-directed mutagenesis, we constructed mutant enzymes in which Arg-46 was replaced by glutamine (R46Q) and alanine (R46A) and examined their kinetic properties. The principal effects in these mutant enzymes were that the Km and Ki values for NADP+ increased by 2 to 3 orders of magnitude over those of the wild-type enzyme. No other kinetic constant was altered more than 6.5-fold. Changing this single amino acid leads to mutant glucose-6-phosphate dehydrogenases with coenzyme specificities that favor NAD+, whereas the wild-type enzyme prefers NADP+ as coenzyme. These results confirm that Arg-46 plays a key role in NADP+ binding by contributing a positively charged planar residue that interacts primarily with the 2'-adenosine phosphate. The Arg residue corresponding to Arg-46 in L. mesenteroides glucose-6-phosphate dehydrogenase is conserved in all glucose-6-phosphate dehydrogenases and, presumably, plays the same role in all these enzymes.