A positively charged residue, R219, was found to interact with the pyridine nitrogen of pyridoxal phosphate in the structure of alanine racemase from Bacillus stearothermophilus [Shaw et al. (1997) Biochemistry 36, 1329- 1342]. Three site-directed mutants, R219K, R219A, and R219E, have been characterized and compared to the wild type enzyme (WT) to investigate the role of R219 in catalysis. The R219K mutation is functionally conservative, retaining ~25% of the WT activity. The R219A and R219E mutations decrease enzyme activity by approximately 100- and 1000-fold, respectively. These results demonstrate that a positively charged residue at this position is required for efficient catalysis. R219 and Y265 are connected through H166 via hydrogen bonds. The R219 mutants exhibit similar kinetic isotope effect trends: increased primary isotope effects (1.5-2-fold) but unchanged solvent isotope effects in the L → D direction and increased solvent isotope effects (1.5-2-fold) but unchanged primary isotope effects in the D → L direction. These results support a two-base racemization mechanism involving Y265 and K39. They additionally suggest that Y265 is selectively perturbed by R219 mutations through the H166 hydrogen-bond network. pH profiles show a large pK(a) shift from 7.1-7.4 (WT and R219K) to 9.5-10.4 (R219A and R219E) for k(cat)/K(M), and from 7.3 to 9.9-10.4 for k(cat). The group responsible for this ionization is likely to be the phenolic hydroxyl of Y265, whose pK(a) is electrostatically perturbed in the WT by the H166-mediated interaction with R219. Accumulation of an absorbance band at 510 nm, indicative of a quinonoid intermediate, only in the D → L direction with R219E provides additional evidence for a two-base mechanism involving Y265.
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