Isolierung und Charakterisierung eines membrangebundenen Pyruvatdehydrogenase-Komplexes aus dem phototrophen Bakterium Rhodospirillum rubrum /Isolation and Characterization of a Membrane-Bound Pyruvate Dehydrogenase Complex from the Phototrophic Bacterium Rhodospirillum rubrum

Abstract

The pyruvate dehydrogenase complex from the photosynthetic bacterium Rhodospirillum rubrum was associated with the membrane fraction both in heterotrophically and photosynthetically grown cells. The complex was separated from the membranes and partially purified by precipitation with MgSO4 and gelfiltration through Sepharose 4B. The purified complex had a specific activity of 1.5-2mumol/min-mg protein and contained the following partial activities: pyruvate dehydrogenase (EC 1.2.4.1), dihydrolipoamide transacetylase (EC 2.3.1.12) and dihydrolipoamide dehydrogenase (EC 1.6.4.3). Contrary to other bacterial pyruvate dehydrogenase complexes, the enzyme complex from R. rubrum revealed no cooperatively between pyruvate binding sites. The kinetic constants (Km) for the overall reaction were (in mM): 0.14 (pyruvate), 0.07 (NAD) and 0.025 (coenzyme A). The Km for thiamine pyrophosphate was dependent on the nature and the concentration of the divalent metal ion (Mn or Mg) present in the reaction mixture, the values ranging from 0.5 to 3 micrometer. NADH was a potent inhibitor (Ki=5 micrometer) of the enzyme complex and the dihydrolipo amide dehydrogenase. The inhibition was competitive with respect to NAD. In addition to its rapid inhibitory effect, NADH also inactivated the enzyme. Cysteine partially protected the enzyme complex against NADH-inactivation. Acetyl-coenzyme A also inhibited the overall reaction (Ki=40 micrometer). The inhibition was dependent on the concentration of coenzyme A, but independent of the concentration of pyruvate. Sugar phosphates, phosphoenolpyruvate, citric acid cycle intermediates and nucleosidephosphates (1 mM) had no pronounced effect on the overall reaction.