Kinetic mechanisms of glycine oxidase from Bacillus subtilis.
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 approximately 3 x 10(4) M(-1) x 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.
Molla
Gianluca
G
Department of Structural and Functional Biology, University of Insubria, Varese, Italy.
Motteran
Laura
L
Job
Viviana
V
Pilone
Mirella S
MS
Pollegioni
Loredano
L
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D011392:Proline / Q000737:chemistry / Q000378:metabolism; D012521:Sarcosine / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011392:Proline; D000594:Amino Acid Oxidoreductases; C115925:glycine oxidase; D010100:Oxygen; D005998:Glycine; D012521:Sarcosine