Kinetic studies of the lipid-activated pyruvate oxidase flavoprotein of Escherichia coli.

摘要: Pyruvate oxidase is a flavoprotein dehydrogenase isolated from Escherichia coli which catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2. In vivo, enzyme can bind bacterial membrane reduce ubiquinone-8, feeding electrons into respiratory chain. The purified has been shown previously phospholipids detergents and, upon doing so, activated. turnover with ferricyanide as an electron acceptor increases 20- 30-fold lipid binding. this work, initial velocity stop-flow kinetics are used investigate activation enzyme. It that unactivated form markedly hysteretic. Progress curves at low substrate concentrations show acceleration in turnover. This consistent results experiments. Rates obtained for either reduction by or its reoxidation single experiments much slower than rates predicted observed studies, some cases more 2 orders magnitude. data best explained slow interconversion between two forms enzyme, one rapidly turns over. As isolated, highly unreactive, revealed During turnover, even absence activators, converts rapid-turnover form. kinetic simulation preclude steady state being established. Lipid activators appear shift equilibrium favor Once "locked" activated conformation, hysteresis no longer observed, agreement Activation appears result both increased transfer out flavin.