Tyrosine 265 of Alanine Racemase Serves as a Base Abstracting -Hydrogen from L-Alanine: The Counterpart Residue to Lysine 39 Specific to D-Alanine

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Abstract
Alanine racemase of Bacillus stearothermophilus has been proposed to catalyze alanine racemization by means of two catalytic bases: lysine 39 (K39) abstracting specifically the α-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. Chenu 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 trans-amination 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'-3H]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 α-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 α-hydrogen of D-alanine to the pro-R position of PMP.

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