[45] d-glucose-6-phosphate dehydrogenases from Pseudomonas fluorescens

D-Glucose 6-phosphate is metabolized in Pseudomonas fluorescens by two pathways, the Entner–Doudoroff pathway and the hexose monophosphate pathway, both starting with the dehydrogenation of D-glucose 6-phosphate. These pathways are assumed to be regulated by two distinct D-glucose-6-phosphate dehydrogenases. The Oglucose-6-phosphate dehydrogenase assigned to the Entner–Doudoroff pathway is subject to complex metabolic regulation by both inductive and allosteric control, whereas no regulatory properties are demonstrated for the dehydrogenase that is a part of the hexose monophosphate pathway. Both D-glucose-6-phosphate dehydrogenases from P. fluorescens show specificity for nicotinamide adenine dinucleotide (NAD+), as well as for nicotinamide adenine dinucleotide phosphate kinase (NADP+), but to a different extent. The assays are based on the spectrophotometric determination of NAD(P)H formed with D-glucose 6-phosphate as a substrate at high pH conditions. The chapter discusses the properties and steps involved in the isolation of Entner–Doudoroff enzyme and Zwischenferment. The isolation procedure for the Entner–Doudoroff enzyme is designed to allow the purification of the Zwischenferment from the same cell-free extract.