Kinetic studies on the role of Lys-171 and Lys-358 in the beta subunit of sarcosine oxidase from Corynebacterium sp. U-96.
Heterotetrameric sarcosine oxidase from Corynebacterium sp.U-96(SO-U96) contains non-covalent and covalent flavins. Lys-358 and Lys-171 in the beta subunit is present at non-covalent flavin adenine dinucleotide (FAD)- and covalent flavin monodinucleotide (FMN)-binding sites, respectively. The Lys-358 mutant, K358R showed 0.07% activity and higher apparent K(m) for sarcosine than the wild-type enzyme, but K358A and K358D mutants showed no activity, suggesting the importance of amino group of Lys358 in the sarcosine-binding to the enzyme. The Lys171 mutants, K171R, K171A and K171D showed 58, 39 and 32% activity of the wild-type enzyme, respectively. An apparent K(m) for oxygen and K(d) of enzyme-sulphite complex increased by the mutation. The rate of reduction of the FAD of K171 mutants with sarcosine did not change by the mutation. The stopped-flow photodiode array analyses of the anaerobic reduction with sarcosin of the wild-type and K171 mutant enzymes showed characteristic spectra of neutral and anionic semiquinones, especially for K171A enzyme. On the basis of these results, the reductive-half reaction of the wild-type and K171 mutant enzymes is explained by a mechanism involving the semiquinones. Low activity of K171 mutants is suggested to be derived from the low rate of oxidation of the reduced FMN in the enzyme.
Saito
Mutsumi
M
Division of Bioscience, Graduate School of Basic Life Science, Kitasato University, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan.
Kanno
Miho
M
Iizuka
Haruo
H
Suzuki
Haruo
H
D003352:Corynebacterium / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004798:Enzymes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008956:Models, Chemical; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012521:Sarcosine / Q000737:chemistry; D050879:Sarcosine Oxidase / Q000737:chemistry*; D013053:Spectrophotometry; D013447:Sulfites / Q000494:pharmacology; D013997:Time Factors
D004798:Enzymes; D013447:Sulfites; D050879:Sarcosine Oxidase; D008239:Lysine; C025026:sodium sulfite; D012521:Sarcosine