Studies on the mechanism of the enzymatic amination and hydration of fumarate.

The finding that the hydration of fumarate in deuterium oxide, as catalyzed by pig heart fumarase, resulted in monodeutero-nmalate, and that prolonged incubations did not lead to any appreciable isotope incorporation into fumarate, established the stcreospecific nature of this reaction (l-3). Through the application of the proton magnetic resonance solid state method, Farrar et al. (4) showed that the protons on carbons 2 and 3 in the monodeutero-r-malate are gauche to one another. On the assumption that the carboxyl groups in crystalline malic acid are trans, the conclusion was reached that fumarase catalyzes a cis addition to the double bond of fumarate. Studies relating to the stereospecificity of enzymatically catalyzed saturation reactions have been restricted to the addition of water to carbon-carbon double bonds (l-3, 5). It was therefore of interest to study reactions in which groups other than water were added to carbon-carbon double bonds. Accordingly, a study was undertaken to delineate some features of the aspartase reaction. This enzyme, catalyzing the reversible deamination of aspartic acid, has been described in a wide variety of microorganisms and its preparation from Bacterium cadaveris in partially purified form has recently been reported (6). The results presented in this paper show that aspartase catalyzes a stereospecific addition of ammonia to the double bond of fumarate. In addition, evidence is presented suggesting that both the aspartase and fumarase from B. cadaveris catalyze a cis addition of ammonia and water, respectively, to the double bond of fumarate.