Flavin‐dependent alcohol oxidase from yeast

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Abstract
The kinetic course of the reaction of methanol and deutero‐methanol with FAD‐dependent alcohol oxidase was investigated under single‐turnover conditions [ked∼ 15000 min−1 (1H3COH) and ∼4300 min−1 (2H3COH)] and multiple‐turnover conditions [TNmax∼6000 min−1 (1H3COH) and ∼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∼ 1.6 mM; kinact∼ 0.55 min−1). In the presence of catalase inactivation during turnover is much slower, the process showing pseudo‐first‐order kinetics (Kinact∼ 0.6 mM; kinact∼ 0.005 min−1 with methanol). The ratio kcat/kinact varies with different alcohols but is always > 105. Propargyl alcohol and methylenecyclopropyl alcohol cannot be considered as suicide substrates, as compared to analogous substrates of other flavin oxidases.