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.
Vought
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Department of Biology, Syracuse University, Syracuse, New York 13244, USA.
Ciccone
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Davino
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Fairbairn
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Lin
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Cosgrove
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Adams
M J
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Levy
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D001120:Arginine / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D019281:Dimerization; D019298:Glucose-6-Phosphate / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011392:Proline; D013816:Thermodynamics
D005973:Glutamine; D009243:NAD; D009249:NADP; D019298:Glucose-6-Phosphate; D001120:Arginine; D011392:Proline; D005954:Glucosephosphate Dehydrogenase; D008239:Lysine