Purification and characterization of the NAD-preferring glucose 6-phosphate dehydrogenase from Acetobacter hansenii (Acetobacter xylinum).
An NAD-preferring glucose 6-phosphate dehydrogenase of Acetobacter hansenii (formerly known as Acetobacter xylinum) has been purified to apparent homogeneity and kinetically characterized. The purified enzyme was stabilized by the use of glycerol, MgSO4, and 2-mercaptoethanol at pH 5.4. The molecular weight of the enzyme, determined by nondenaturing gel filtration, is 243,000. The subunit molecular weight is 60,140 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. At pH 5.4 the enzyme has Kms of 0.104 and 0.34 mM for NAD+ and NADP+, respectively; the Kms for glucose 6-phosphate are 0.071 and 0.089 mM, using NAD+ and NADP+, respectively; and the kcat values are 128,000 and 77,300 min-1 with NAD+ and NADP+, respectively. The Kms for NADP+ and glucose 6-phosphate are approximately 10 times higher than the corresponding Kms for the NADP-specific glucose 6-phosphate dehydrogenase in the same organism, but the kcat is also approximately 10-fold higher, so that the kcat/Km values for these two activities are nearly identical at pH 5.4. Both the NAD- and NADP-linked activities of the NAD-preferring enzyme are inhibited by ATP. The NADP-specific glucose 6-phosphate dehydrogenase is insensitive to ATP at pH 6.7 and 9.5, but at pH 5.4 ATP inhibits this enzyme. The possible roles of these two glucose 6-phosphate dehydrogenases in the metabolism of A. hansenii are discussed.
Ragunathan
S
S
Department of Biology, Syracuse University, New York 13244.
Levy
H R
HR
D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D041822:Gluconacetobacter xylinus / Q000201:enzymology* / Q000254:growth & development; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity
D009243:NAD; D009249:NADP; D005954:Glucosephosphate Dehydrogenase