Purification and properties of NADP-linked glucose-6-phosphate dehydrogenase from Acetobacter hansenii (Acetobacter xylinum).
The NADP-linked glucose-6-phosphate dehydrogenase from Acetobacter hansenii (formerly known as Acetobacter xylinum) has been purified to apparent homogeneity. The sequence of the 10 N-terminal amino acids was determined. The subunit molecular weight of the enzyme is 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; gel filtration studies under nondenaturing conditions revealed that the molecular weight of the enzyme is 200,000 to 220,000 at pH 6.5 and 9.5, suggesting that the native enzyme is a tetramer. Specificity studies at both pH 6.5 and 9.5 demonstrated that the enzyme is a typical NADP-preferring glucose-6-phosphate dehydrogenase. The enzyme's catalytic activity increases with increasing pH, kcat being approximately 4 times greater at pH 9.5 than at pH 6.7 and the Km for NADP+ being 3 times lower at the higher pH; but the Km for glucose 6-phosphate is nearly 20 times higher at pH 9.5 than at pH 6.7, suggesting that the enzyme is catalytically more efficient at the lower pH. At pH 6.7, initial velocity measurements, product inhibition by NADPH, and inhibition by glucosamine 6-phosphate yielded results that were consistent with a steady-state random mechanism. At pH 9.5, steady-state kinetic analyses suggested that the mechanism is ordered, with coenzyme binding first, but nonlinear double-reciprocal plots were observed in the presence of NADPH when glucose 6-phosphate was varied and a complete kinetic analysis was not undertaken. Among several nucleotides and potential inhibitory ligands examined, only 2',5'-ADP inhibited the enzyme significantly.
Levy
H R
HR
Department of Biology, Syracuse University, New York 13244.
Cook
C
C
D000595:Amino Acid Sequence; D003067:Coenzymes / Q000378:metabolism; D004795:Enzyme Stability; D041822:Gluconacetobacter xylinus / Q000201:enzymology*; D005954:Glucosephosphate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism*; D013379:Substrate Specificity
D003067:Coenzymes; D008024:Ligands; D009249:NADP; D005954:Glucosephosphate Dehydrogenase