Flavin-dependent alcohol oxidase from yeast. Studies on the catalytic mechanism and inactivation during turnover.
The kinetic course of the reaction of methanol and deutero-methanol with FAD-dependent alcohol oxidase was investigated under single-turnover conditions [kred approximately equal to 15000 min-1 (1H3COH) and approximately equal to 4300 min-1 (2H3COH)] and multiple-turnover conditions [TNmax approximately equal to 6000 min-1 (1H3COH) and approximately equal to 3100 min-1 (2H3COH)]. A kinetic scheme for the overall catalytic mechanism is proposed, which is characterized by (1) formation of a Michaelis complex between enzyme and substrate, (2) the reductive step involving partly rate-limiting scission of the substrate C-H bond, (3) reaction of the complex of reduced enzyme and aldehyde with dioxygen, and (4) a significant contribution of the dissociation rate of product from its complex with reoxidized enzyme to the overall rate. Prolonged turnover of various alcohols, including methanol, results in progressive inactivation of the enzyme by two processes. In the absence of catalase the inactivation rate increases with time due to accumulation of hydrogen peroxide, which is a potent inactivator (Kd approximately equal to 1.6 mM; kinact approximately equal to 0.55 min-1). In the presence of catalase inactivation during turnover is much slower, the process showing pseudo-first-order kinetics (Kinact approximately equal to 0.6 mM; kinact approximately equal to 0.005 min-1 with methanol). The ratio kcat/kinact varies with different alcohols but is always greater than 10(5). Propargyl alcohol and methylenecyclopropyl alcohol cannot be considered as suicide substrates, as compared to analogous substrates of other flavin oxidases.
D000332:Aerobiosis; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000693:Anaerobiosis; D002175:Candida / Q000201:enzymology*; D002374:Catalase / Q000378:metabolism; D002384:Catalysis; D003903:Deuterium; D005415:Flavins / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D000432:Methanol / Q000378:metabolism / Q000494:pharmacology; D008956:Models, Chemical; D013379:Substrate Specificity
D005415:Flavins; D003903:Deuterium; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D002374:Catalase; D000432:Methanol