On the mechanism of the reaction catalyzed by glucose 6-phosphate dehydrogenase.
The catalytic mechanism of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides was investigated by replacing three amino acids, His-240, Asp-177, and His 178, with asparagine, using site-directed mutagenesis. Each of the mutant enzymes was purified to homogeneity and characterized by substrate binding studies and steady-state kinetic analyses. The three-dimensional structure of the H240N glucose 6-phosphate dehydrogenase was determined at 2.5 A resolution. The results support a mechanism in which His-240 acts as the general base that abstracts the proton from the C1-hydroxyl group of glucose 6-phosphate, and the carboxylate group of Asp-177 stabilizes the positive charge that forms on His-240 in the transition state. The results also confirm the postulated role of His-178 in binding the phosphate moiety of glucose 6-phosphate.
Cosgrove
M S
MS
Department of Biology, Syracuse University, Syracuse, New York 13244, USA.
Naylor
C
C
Paludan
S
S
Adams
M J
MJ
Levy
H R
HR
D001224:Aspartic Acid / Q000378:metabolism; D002384:Catalysis; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D007934:Leuconostoc / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D001224:Aspartic Acid; D006639:Histidine; D005954:Glucosephosphate Dehydrogenase