Membrane-Bound D-Gluconate Dehydrogenase from Pseudomonas aeruginosaIts Kinetic Properties and a Reconstitution of Gluconate Oxidase

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Abstract
D-Gluconate dehydrogenase solubilized and purified from the membrane of Pseudomonas aeruginosa showed a Km value that varied with pH, namely 0.8 mM at pH 6.0 and 3.2 mM at pH 5.0. The enzyme was highly specific for D-gluconate and had an optimum pH 5.0–6.0. These kinetic properties of the solubilized enzyme were basically the same as those of the native enzyme in the membrane. The enzyme activity was inhibited competitively by pyruvate and 2-ketogluconate, non-competitively by oxamate and in a mixed-type fashion by oxalate. The enzyme could reduce phenazine methosulfate, 2,6-dichlorophenolindophenol, ferricyanide, and coenzyme Q1, but not menadione. D-Gluconate oxidase activity was restored by addition of coenzyme Q2 to a mixture of the purified enzyme and the enzyme-depleted membrane. Coenzyme Q1 or Q4 was also effective to a lesser degree. Coenzyme Q2 appeared to mediate between the enzyme and the membrane residue in an electron transport pathway in which 2,6-dichlorophenolindophenol or tetramethylphenylenediamine was not involved. The oxidase activity restored by adding coenzyme Q2 was inhibited by cyanide, azide, antimycin A, and n-heptyl-4-hydroxyquinoline-N-oxide. The reconstituted oxidase system possessed a higher optimum pH than the native oxidase system. Based on these results, the function of each of the three polypeptide components of purified gluconate dehydrogenase is discussed in relation to electron transport in gluconate oxidation.

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