Biospecific inactivation of aspartase by L-aspartic-β-semialdehyde

Abstract Aspartase purified from Escherichia coli W cells was rapidly and irreversibly inactivated by L-aspartic-β-semialdehyde (ASA), a substrate analog, following pseudo-first order kinetics. The inactivation rate showed a tendency to saturate as the ASA concentration increased. The increase in pH and the addition of Mg 2+ at the alkaline pH accelerated the inactivation. In addition to chemically synthesized ASA, modification of aspartase by enzymatically generated ASA was attempted. Since the reaction equilibrium of homoserine dehydrogenase is extremely unfavorable for ASA formation, glutamate dehydrogenase reaction was coupled to it. When aspartase was incubated with these two enzyme systems, a time-dependent inactivation was observed. L-Aspartate, a substrate for the enzyme, protected it from inactivation. Analysis of the sulfhydryl group indicated that among 9 sulfhydryl groups per enzyme subunit, one residue essential for the activity was involved in the ASA-mediated inactivation.