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 h ave been characterized by steady-state kinetic analysis. Phenazine eth osulfate, a commonly used electron acceptor for this enzyme, was shown to exhibit pronounced substrate inhibition with a K(i) value approxim ately 20-fold lower than its K(m). Wurster's Blue exhibited only relat ively mild substrate inhibition and was deemed a more appropriate elec tron acceptor. Ammonia was an obligatory activator of the enzyme at lo w concentrations and inhibited a high concentrations. The K(i) value f or this inhibition correlated closely with the K(d) calculated from a titration of perturbations of the absorption spectrum of methanol dehy drogenase 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 com petitive inhibitor with respect to methanol. Kinetic analysis indicate d that the latter two activities corresponded to two distinct binding sites for cyanide. The K(a) for cyanide activation correlated closely with the concentration required to inhibit 50% of the endogenous react ion, suggesting that a single binding event is responsible for both of these effects. A model is presented to describe the effects of ammoni a and cyanide in the reaction cycle of methanol dehydrogenase, and the physiological relevance of the activation and inhibition by these com pounds in vitro is discussed.