Kinetics of Thermostable Alanine Racemase of Bacillus stearothermophilus

Unlabeled D- and L-alanine were racemized in deuterium oxide with an alanine racemase of Bacillus stearothermophilus at saturated concentration of substrate, and various p²H and temperature. Samples of the solution were taken at intervals, and all alanine isomers in the samples were transformed into a mixture of diastereomeric derivatives of methyl N-(–)-camphanylalaninate. Their ratio was measured on a GC-Mass, and the relative rate was calculated at the initial stage of the reaction. There was little difference in the decrease rate of the optical rotation between the enantiomers. Internal proton-transfer to the antipode was almost zero for either substrate. The α-hydrogen was abstracted 1.2–2.3 times faster from D-alanine than from L-alanine. D-Alanine gave an almost even mixture of deuterium labeled D- and L-alanine, while L-alanine gave a mixture of labeled D- and L-alanine at a ratio of 3:1. These results suggest the racemase builds two different bases in the active site. The base for D-alanine may be closer to the enzyme surface, and that for L-alanine inside.