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. 1973 Dec;116(3):1150–1159. doi: 10.1128/jb.116.3.1150-1159.1973

Purification and Regulation of Glucose-6-Phosphate Dehydrogenase from Bacillus licheniformis

Dennis Opheim 1, R W Bernlohr 1
PMCID: PMC246469  PMID: 4148096

Abstract

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 Km of approximately 5 μM 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+ (Km = 10 μM). Phosphoenolpyruvate (Km = 1.6 mM), adenosine 5′-triphosphate (Km = 0.5 mM), adenosine diphosphate (Km = 1.5 mM), and adenosine 5′-monophosphate (Km = 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 × 105. Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is composed of two similar subunits of approximately 6 × 104 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.

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Selected References

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