title: Role of tyrosine 265 of alanine racemase from Bacillus stearothermophilus.
abstract: Tyrosine 265 (Y265) of Bacillus stearothermophilus is believed to serve as a catalytic base specific to the L-enantiomer of a substrate amino acid by removing (or returning) an alpha-hydrogen from (or to) the isomer on the basis of the X-ray structure of the enzyme [Stamper, C.G., Morollo, A.A., and Ringe, D. (1998) Biochemistry 37, 10438-10443]. We found that the Y265-->Ala mutant (Y265A) enzyme is virtually inactive as a catalyst for alanine racemization. We examined the role of Y265 further with beta-chloroalanine as a substrate with the expectation that the Y265A mutant only catalyzes the alpha,beta-elimination of the D-enantiomer of beta-chloroalanine. However, L-beta-chloroalanine also served as a substrate; this enantiomer was rather better as a substrate than its antipode. Moreover, the mutant enzyme was as equally active as the wild-type enzyme in the elimination reaction. These findings indicate that Y265 is essential for alanine racemization but not for beta-chloroalanine elimination.
authors.affiliation: Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Watanabe
authors.forename: Y
authors.initials: Y
authors.lastname: Kurokawa
authors.forename: T
authors.initials: T
authors.lastname: Yoshimura
authors.forename: N
authors.initials: N
authors.lastname: Esaki
mesh_terms: D000411:Alanine Racemase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D013237:Stereoisomerism; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D015091:beta-Alanine; C006977:3-chloroalanine; D014443:Tyrosine; D000411:Alanine Racemase