An examination of the role of asp-177 in the His-Asp catalytic dyad of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: X-ray structure and pH dependence of kinetic parameters of the D177N mutant enzyme.
The role of Asp-177 in the His-Asp catalytic dyad of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides has been investigated by a structural and functional characterization of the D177N mutant enzyme. Its three-dimensional structure has been determined by X-ray cryocrystallography in the presence of NAD(+) and in the presence of glucose 6-phosphate plus NADPH. The structure of a glucose 6-phosphate complex of a mutant (Q365C) with normal enzyme activity has also been determined and substrate binding compared. To understand the effect of Asp-177 on the ionization properties of the catalytic base His-240, the pH dependence of kinetic parameters has been determined for the D177N mutant and compared to that of the wild-type enzyme. The structures give details of glucose 6-phosphate binding and show that replacement of the Asp-177 of the catalytic dyad with asparagine does not affect the overall structure of glucose 6-phosphate dehydrogenase. Additionally, the evidence suggests that the productive tautomer of His-240 in the D177N mutant enzyme is stabilized by a hydrogen bond with Asn-177; hence, the mutation does not affect tautomer stabilization. We conclude, therefore, that the absence of a negatively charged aspartate at 177 accounts for the decrease in catalytic activity at pH 7.8. Structural analysis suggests that the pH dependence of the kinetic parameters of D177N glucose 6-phosphate dehydrogenase results from an ionized water molecule replacing the missing negative charge of the mutated Asp-177 at high pH. Glucose 6-phosphate binding orders and orients His-178 in the D177N-glucose 6-phosphate-NADPH ternary complex and appears to be necessary to form this water-binding site.
Cosgrove
M S
MS
Department of Biology, Syracuse University, Syracuse, New York 13244, USA.
Gover
S
S
Naylor
C E
CE
Vandeputte-Rutten
L
L
Adams
M J
MJ
Levy
H R
HR
D001224:Aspartic Acid / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019298:Glucose-6-Phosphate / Q000737:chemistry; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D008956:Models, Chemical; D008958:Models, Molecular; D009068:Movement; D016297:Mutagenesis, Site-Directed; D009154:Mutation
D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D019298:Glucose-6-Phosphate; D005954:Glucosephosphate Dehydrogenase