Purification and regulation of glucose-6-phosphate dehydrogenase from Bacillus licheniformis.
d-Glucose-6-phosphate nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase (EC 1.1.1.49) from Bacillus licheniformis has been purified approximately 600-fold. The enzyme appears to be constitutive and exhibits activity with either oxidized NAD (NAD(+)) or oxidized NADP (NADP(+)) as electron acceptor. The enzyme has a pH optimum of 9.0 and has an absolute requirement for cations, either monovalent or divalent. The enzyme exhibits a K(m) of approximately 5 muM for NADP(+), 3 mM for NAD(+), and 0.2 mM for glucose-6-phosphate. Reduced NADP (NADPH) is a competitive inhibitor with respect to NADP(+) (K(m) = 10 muM). Phosphoenolpyruvate (K(m) = 1.6 mM), adenosine 5'-triphosphate (K(m) = 0.5 mM), adenosine diphosphate (K(m) = 1.5 mM), and adenosine 5'-monophosphate (K(m) = 3.0 mM) are competitive inhibitors with respect to NAD(+). The molecular weight as estimated from sucrose density centrifugation and molecular sieve chromatography is 1.1 x 10(5). Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is composed of two similar subunits of approximately 6 x 10(4) molecular weight. The intracellular levels of glucose-6-phosphate, NAD(+), and NADP(+) were measured and found to be approximately 1 mM, 0.9 mM, and 0.2 mM, respectively, during logarithmic growth. From a consideration of the substrate pool sizes and types of inhibitors, we conclude that this single constitutive enzyme may function in two roles in the cell-NADH production for energetics and NADPH production for reductive biosynthesis.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000645:Ammonium Sulfate; D001407:Bacillus / Q000201:enzymology* / Q000378:metabolism; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D004589:Electrophoresis, Disc; D004950:Esterases / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D010734:Phosphogluconate Dehydrogenase / Q000378:metabolism; D014461:Ultracentrifugation
D009243:NAD; D000249:Adenosine Monophosphate; D009249:NADP; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D010734:Phosphogluconate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D004950:Esterases; D000645:Ammonium Sulfate