Kinetic studies on the reaction mechanism of sarcosine oxidase.
A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC 1.5.3.1) isolated from Corynebacterium sp. U-96 contains both covalently bound FAD and noncovalently bound FAD. The noncovalent FAD reacts with sarcosine, the covalent FAD with molecular oxygen (Jorns, M.S. (1985) Biochemistry 24, 3189-3194). To clarify the reaction mechanism of the enzyme, kinetic investigations were performed by the stopped-flow method as well as by analysis of the overall reaction. The absorption spectrum of the enzyme in the steady state was very similar to that of the oxidized enzyme, and no intermediate enzyme species, such as a semiquinoid flavin, was detected. The rate for anaerobic reduction of the noncovalently bound FAD and the covalently bound FAD by sarcosine were 31 and 6.7 s-1, respectively. The latter value was smaller than the value of respective Vmax/e0 obtained by the overall reaction kinetics (Vmax/e0: the maximum velocity per enzyme concentration). Both rate constants for oxidation of the two FADs by molecular oxygen were 100 s-1. A reaction scheme of sarcosine oxidase is proposed to account for the data obtained; 70% of the enzyme functions via a fully reduced enzyme, and 30% of the enzyme goes along a side-path, without forming the fully reduced enzyme. In addition, it is suggested that the reactivity of noncovalently bound FAD with sarcosine is affected by the oxidation-reduction state of the covalently bound FAD, in contrast to the reactivity of the covalently bound FAD with molecular oxygen, which is independent of the oxidation-reduction state of the noncovalently bound FAD.
D000693:Anaerobiosis; D003352:Corynebacterium / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D012521:Sarcosine / Q000378:metabolism; D050879:Sarcosine Oxidase; D013053:Spectrophotometry
D005182:Flavin-Adenine Dinucleotide; D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase; D010100:Oxygen; D012521:Sarcosine