The Kinetics of Reoxidation of Reduced Benzylamine Oxidase

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Abstract
1 The mechanism of reoxidation of reduced benzylamine oxidase has been investigated at different pH between 6 and 10 by steady-state and transient-state kinetic methods. 2 The reoxidation process involves minimally a second-order interaction between reduced enzyme and oxygen leading to the formation of a spectrally modified enzyme intermediate, and a subsequent first-order step converting this intermediate into free enzyme. The variation with pH of rate constants according to such a reaction scheme is reported. 3 Under aerobic conditions the oxygen-independent reaction represents the main rate-limiting step in the catalytic process at alkaline pH. At neutral or acid pH the interaction between reduced enzyme and oxygen becomes mainly rate-limiting, indicating that the concentration of oxygen may be a critical factor controlling enzyme activity under physiological conditions. 4 The spectrally modified intermediate formed during the reoxidation process exhibits a dif-ference-absorption band centered around 290 nm in comparison to free enzyme, and an additional difference-absorption band at 470 nm in comparison to reduced enzyme. These data indicate that formation of the intermediate, besides leading to a reappearance of the 470-nm absorption band dis-appearing on reduction of the enzyme, results in a spectral perturbation of one or several aromatic amino-acid residues in the protein. This perturbation could possibly reflect a conformational change of the enzyme.