Tyrosine 265 of alanine racemase serves as a base abstracting alpha-hydrogen from L-alanine: the counterpart residue to lysine 39 specific to D-alanine.
Alanine racemase of Bacillus stearothermophilus has been proposed to catalyze alanine racemization by means of two catalytic bases: lysine 39 (K39) abstracting specifically the alpha-hydrogen of D-alanine and tyrosine 265 (Y265) playing the corresponding role for the antipode L-alanine. The role of K39 as indicated has already been verified [Watanabe, A., Kurokawa, Y., Yoshimura, T., Kurihara, T., Soda, K., and Esaki, N. (1999) J. Biol. Chem. 274, 4189-4194]. We here present evidence for the functioning of Y265 as the base catalyst specific to L-alanine. The Y265-->Ala mutant enzyme (Y265A), like Y265S and Y265F, was a poor catalyst for alanine racemization. However, Y265A and Y265S catalyzed transamination with D-alanine much more rapidly than the wild-type enzyme, and the bound coenzyme, pyridoxal 5'-phosphate (PLP), was converted to pyridoxamine 5'-phosphate (PMP). The rate of transamination catalyzed by Y265F was about 9% of that by the wild-type enzyme. However, Y265A, Y265S, and Y265F were similar in that L-alanine was inert as a substrate in transamination. The apo-form of the wild-type enzyme catalyzes the abstraction of tritium non-specifically from both (4'S)- and (4'R)-[4'-(3)H]PMP in the presence of pyruvate. In contrast, apo-Y265A abstracts tritium virtually from only the R-isomer. This indicates that the side-chain of Y265 abstracts the alpha-hydrogen of L-alanine and transfers it supra-facially to the pro-S position at C-4' of PMP. Y265 is the counterpart residue to K39 that transfers the alpha-hydrogen of D-alanine to the pro-R position of PMP.
Watanabe
A
A
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
Yoshimura
T
T
Mikami
B
B
Esaki
N
N
D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000411:Alanine Racemase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D013237:Stereoisomerism; D014443:Tyrosine / Q000737:chemistry
D017931:DNA Primers; D014443:Tyrosine; D000411:Alanine Racemase; D008239:Lysine; D000409:Alanine