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Journal of Biochemistry Advance Access published online on March 29, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm084
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© 2007 The Japanese Biochemical Society

KINETIC STUDIES ON THE ROLE OF Lys-171 AND Lys-358 IN THE ß SUBUNIT OF SARCOSINE OXIDASE FROM Corynebacterium sp. U-96.

Mutsumi Saito1, Miho Kanno1, Haruo Iizuka2 and Haruo Suzuki1,2

1Division of Bioscience, Graduate School of Basic Life Science, and 2Department of Biosciences, School of Science, Kitasato University, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan

All correspondence to: Haruo Suzuki, Department of Biosciences, School of Science, Kitasato University, Kitasato 1-15-1, Sagamihara-shi, Kanagawa 228-8555, Japan, Tel and Fax: +81-48-778-9410, E-mail: suzukih{at}kitasato-u.ac.jp

Received January 30, 2007; Accepted March 21, 2007


  Abstract

Heterotetrameric sarcosine oxidase from Corynebacterium sp.U-96(SO-U96) contains noncovalent and covalent flavins. Lys-358 and Lys-171 in the ß subunit is present at noncovalent FAD- and covalent FMN-binding sites, respectively. The Lys-358 mutant, K358R showed 0.07% activity and higher apparent Km 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 Km for oxygen and Kd of enzyme-sulfite 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.

Key Words: flavoenzyme, kinetic mechanism, essential Lys, sarcosine oxidase, site-directed mutagenesis


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