Aspartate Transcarbamylase

Abstract

Abstract N-(Phosphonacetyl)-l-aspartate (PALA) is a stable analogue of the transition state in the reaction catalyzed by aspartate transcarbamylase from Escherichia coli; it combines in 1 molecule most of the structural features of the two natural substrates, carbamyl phosphate and l-aspartate. The inhibition constant (Ki) for PALA with the catalytic subunit is 2.7 x 10-8m at 28° and pH 7.0 in 0.2 m imidazole acetate; the value of Ki is approximately the same with the native enzyme. PALA binds to aspartate transcarbamylase about 1000 times more tightly than carbamyl phosphate, the substrate bound most tightly. In addition to tight binding, other data indicate that PALA interacts with the enzyme at both the carbamyl phosphate and l-aspartate sites simultaneously and puts the enzyme into the same contracted conformation as do carbamyl phosphate and succinate (an analogue of l-aspartate) acting together. PALA alone or carbamyl phosphate and succinate together produce the same ultraviolet difference spectrum with the catalytic subunit; the binding of PALA alone or carbamyl phosphate and succinate together at some of the catalytic sites in the native enzyme activates the remaining catalytic sites to the same extent. PALA gives competitive inhibition with carbamyl phosphate but not with l-aspartate, indicating an obligatory binding order for the substrates: carbamyl phosphate must bind before l-aspartate for catalysis to occur. Titration of the catalytic subunit with PALA, monitored by ultraviolet difference spectroscopy, indicates that there are 3.07 active sites per 100,000 daltons. The interaction of PALA with the enzyme is consistent with the compression model for aspartate transcarbamylase previously proposed (Collins, K.D., and Stark, G.R., J. Biol. Chem., 244, 1869 (1969)).