Ligand interactions at the active site of aspartate transcarbamoylase from Escherichia coli.

The active site of aspartate transcarbamoylase from Escherichia coli was probed by studying the inhibitory effects of substrate analogues on the catalytic subunit of the enzyme. The inhibitors were chosen to satisfy the structural requirements for binding to either the phosphate or the dicarboxylate region. In addition, they also contained a side chain that would extend into the normal position occupied by the carbamoyl group. All the compounds tested showed competitive inhibition against carbamoyl phosphate. The ionic character of the side chain was found to be highly important in determining the affinity of the inhibitor. On the other hand, very little effect on binding was produced by changing the geometry of the functional group from trigonal to tetrahedral. Our findings suggest that the electrostatic stabilization of the negative charge that develops in the transition state may be a major factor in promoting catalysis. From the available X-ray diffraction data, we propose His-134 as the residue most likely to participate in this interaction. These results have significant implications on the design of reversible and irreversible inhibitors to this enzyme.