Mechanism of alpha-amino-epsilon-caprolactam racemase reaction.

Abstract

alpha-Amino-epsilon-caprolactam racemase catalyzes the exchange of the alpha-hydrogen of the substrate with deuterium during racemization in deuterium oxide. The rate of the hydrogen exchange measured by 1H NMR is lower than that of racemization in deuterium oxide for both the enantiomers. Both the enantiomers of alpha-amino-epsilon-caprolactam show an overshoot of the optical rotation during the enzymatic racemization in deuterium oxide (but not in water). This phenomenon may be attributable to a primary deuterium isotope effect at the alpha-position: alpha-deuterium isotope effects of 3.6 and 2.0 were observed for the racemization of the D and L enantiomers of alpha-amino-epsilon-caprolactam, respectively. Results of tritium-labeling experiments showed that the enzyme catalyzes both retention and inversion of configuration of the substrate with a similar probability in each turnover. Conversion of [alpha-2H]-D-alpha-amino-epsilon-caprolactam in water and unlabeled D-alpha-amino-epsilon-caprolactam in deuterium oxide into the L isomer under nearly single turnover conditions with the enzyme showed significant internal return of the alpha-hydrogen. These results support a single base mechanism for the racemization reaction catalyzed by the enzyme.