A new kinetic model for the steady-state reactions of the quinoprotein methanol dehydrogenase from Paracoccus denitrificans.
The reactions of methanol dehydrogenase from Paracoccus denitrificans with artificial electron acceptors, ammonia, cyanide, and substrates have been characterized by steady-state kinetic analysis. Phenazine ethosulfate, a commonly used electron acceptor for this enzyme, was shown to exhibit pronounced substrate inhibition with a K(i) value approximately 20-fold lower than its Km. Wurster's Blue exhibited only relatively mild substrate inhibition and was deemed a more appropriate electron acceptor. Ammonia was an obligatory activator of the enzyme at low concentrations and inhibited a high concentrations. The K(i) value for this inhibition correlated closely with the Kd calculated from a titration of perturbations of the absorption spectrum of methanol dehydrogenase which were caused by the addition of ammonia. Cyanide, which suppressed the peculiar endogenous reaction of methanol dehydrogenase, was also both an activator of substrate-dependent activity and a competitive inhibitor with respect to methanol. Kinetic analysis indicated that the latter two activities corresponded to two distinct binding sites for cyanide. The Ka for cyanide activation correlated closely with the concentration required to inhibit 50% of the endogenous reaction, suggesting that a single binding event is responsible for both of these effects. A model is presented to describe the effects of ammonia and cyanide in the reaction cycle of methanol dehydrogenase, and the physiological relevance of the activation and inhibition by these compounds in vitro is discussed.
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
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Harris
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Davidson
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D045542:PQQ Cofactor; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011190:Potassium Cyanide / Q000494:pharmacology; D015363:Quinolones / Q000378:metabolism; D013379:Substrate Specificity
D003067:Coenzymes; D015363:Quinolones; D045542:PQQ Cofactor; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D011190:Potassium Cyanide