Catalytic and redox properties of glycine oxidase from Bacillus subtilis.
Glycine oxidase (GO) from Bacillus subtilis is a homotetrameric flavoprotein oxidase that catalyzes the oxidation of the amine functional group of sarcosine or glycine (and some D-amino acids) to yield the corresponding keto acids, ammonia/amine and H(2)O(2). It shows optima at pH 7-8 for stability and pH 9-10 for activity, depending on the substrate. The tetrameric oligomeric state of the holoenzyme is not affected by pH in the 6.5-10 range. Free GO forms the anionic red semiquinone upon photoreduction. This species is thermodynamically stable, as indicated by the large separation of the two single-electron reduction potentials (DeltaE >or = 290 mV). The first potential is pH independent, while the second is dependent. The midpoint reduction potential exhibits a -23.4 mV/pH unit slope, which is consistent with an overall two-electrons/one-proton transfer in the reduction to yield anionic reduced flavin. In the presence of glycolate (a substrate analogue) and at pH 7.5 the potential for the semiquinone-reduced enzyme couple is shifted positively by approximately 160 mV: this favors a two-electron transfer compared to the free enzyme. Binding of glycolate and sulfite is also affected by pH, showing dependencies that reflect the ionization of an active site residue with a pK(a) approximately equal 8.0. These results highlight substantial differences between GO and related flavoenzymes. This knowledge will facilitate biotechnological use of GO, e.g. as an innovative tool for the in vivo detection of the neurotransmitter glycine.
Pedotti
Mattia
M
Department of Biotechnology and Molecular Sciences, University of Insubria, Varese, Italy.
Ghisla
Sandro
S
Motteran
Laura
L
Molla
Gianluca
G
Pollegioni
Loredano
L
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism*; D016227:Benzoquinones / Q000378:metabolism; D004795:Enzyme Stability; D006016:Glycolates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013447:Sulfites / Q000378:metabolism
D001426:Bacterial Proteins; D016227:Benzoquinones; D006016:Glycolates; D013447:Sulfites; C031149:glycolic acid; C025232:semiquinone radicals; D000594:Amino Acid Oxidoreductases; C115925:glycine oxidase