Membrane-boundd-Gluconate Dehydrogenase ofSerratia marcescens: Purification and Properties

Abstract

Membrane-bound d-gluconate dehydrogenase, which catalyzes oxidation of d-gluconate to 2-keto-d-gluconate in the presence of an electron transport system, was purified to homogeneous state from Serratia marcescens IFO 3054. Purification was achieved at about 450-fold with an overall yield of 18% from the particulate fraction by solubilization with Triton X-100, subsequent fractionations on DEAE-cellulose and DEAE-Sephadex column chromatographies, and precipitation with polyethylene glycol 6000. The purified enzyme preparation was found to be an enzyme-cytochrome complex and the dehydrogenase protein tightly bound cytochrome c₁ component which had the absorption maxima at 418, 523 and 554 nm. The substrate specificity of the enzyme seemed to be restricted to only d-gluconate and the apparent Km value of the purified enzyme for d-gluconate was 0.8 × 10^-3 m. Enzyme activity was inhibited by pyruvate competitively and by oxamate noncompetitively.