Studies on the mechanism of action of methoxatin-requiring methanol dehydrogenase: reaction of enzyme with electron-acceptor dye.
Bacterial methoxatin-dependent methanol dehydrogenase requires an electron acceptor, e.g., phenazine methosulfate (PMS), for activity. Oxidation of methanol shows a deuterium isotope effect of 4.3 in the presence of high concentrations of PMS; the effect is reduced at low PMS concentrations. This suggests that the catalytic reaction comprises at least two steps, one involving substrate and the other PMS. The UV-visible spectrum of methanol dehydrogenase undergoes a series of characteristic changes when PMS is added to the enzyme. It is proposed that these changes correspond to the formation of an enzyme intermediate, resulting from the reaction of PMS with the enzyme, which can subsequently react with substrate. Preincubation of the enzyme with PMS followed by dilution into a solution containing [14C]methanol results in an additional enzyme turnover compared to the control performed without preincubation with PMS. This confirms the proposal that the enzyme reacts with PMS before reacting with substrate. The reaction between the enzyme and PMS is not a redox reaction since no oxygen is consumed during the reaction nor is the PMS reduced. Methanol dehydrogenase is inactivated by cyclopropanol in the presence of PMS. The intermediate formed by reaction of the enzyme with PMS is also an intermediate in the cyclopropanol inactivation reaction. Preincubation of the enzyme with PMS gives an increase in the rate of inactivation by cyclopropanol. Cyclopropanol stoichiometrically equivalent to 13% of the enzyme is sufficient to completely inactivate the enzyme. The inactivation reaction shows neither a deuterium nor a tritium isotope effect.(ABSTRACT TRUNCATED AT 250 WORDS)
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism; D008772:Methylococcaceae / Q000378:metabolism; D008773:Methylphenazonium Methosulfate; D045542:PQQ Cofactor; D011804:Quinolines / Q000378:metabolism*
D003067:Coenzymes; D011804:Quinolines; D008773:Methylphenazonium Methosulfate; D045542:PQQ Cofactor; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor)