Abstract
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.
