Kinetic mechanisms of glycine oxidase from Bacillus subtilis

Abstract

The kinetic properties of glycine oxidase from Bacillus subtilis were investigated using glycine, sarcosine, and D-proline as substrate. The turnover numbers at saturating substrate and oxygen concentrations were 4.0 s-1, 4.2 s-1, and 3.5 s-1, respectively, with glycine, sarcosine, and D-proline as substrate. Glycine oxidase was converted to a two-electron reduced form upon anaerobic reduction with the individual substrates and its reductive half-reaction was demonstrated to be reversible. The rates of flavin reduction extrapolated to saturating substrate concentration, and under anaerobic conditions, were 166 s-1, 170 s-1, and 26 s-1, respectively, with glycine, sarcosine, and D-proline as substrate. The rate of reoxidation of reduced glycine oxidase with oxygen in the absence of product (extrapolated rate ≈ 3 × 104 M-1·S-1) was too slow to account for catalysis and thus reoxidation started from the reduced enzyme:imino acid complex. The kinetic data are compatible with a ternary complex sequential mechanism in which the rate of product dissociation from the reoxidized enzyme form represents the rate-limiting step. Although glycine oxidase and D-amino acid oxidase differ in substrate specificity and amino acid sequence, the kinetic mechanism of glycine oxidase is similar to that determined for mammalian D-amino acid oxidase on neutral D-amino acids, further supporting a close similarity between these two amine oxidases.