The regulatory subunit of Escherichia coli aspartate carbamoyltransferase may influence homotropic cooperativity and heterotropic interactions by a direct interaction with the loop containing residues 230-245 of the catalytic chain.

Abstract

A recent x-ray structure of aspartate carbamoyltransferase (carbamoyl-phosphate: L-aspartate carbamoyl-transferase, EC 2.1.3.2) with phosphonoacetamide bound [Gouaux, J. E. & Lipscomb, W. N. (1990) Biochemistry 29, 389-402] shows an interaction between Asp-236 of the catalytic chain and Lys-143 of the regulatory chain. Asp-236 is part of the loop containing residues 230-245 (240s) of the catalytic chain that undergoes a significant conformational change between the tight and the relaxed states of the enzyme. Furthermore, side-chain interactions between the 240s loop and other portions of the enzyme have been shown to be important for the low activity and low affinity of the tight state and the high activity and high affinity of the relaxed state. To determine whether the intersubunit link between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain is important for either homotropic cooperativity and/or the heterotropic interactions in aspartate carbamoyltransferase, site-specific mutagenesis was used to replace Asp-236 with alanine. The mutant enzyme exhibits full activity and a loss of both homotropic cooperativity and heterotropic interactions. Furthermore, the aspartate concentration at half the maximal observed specific activity is reduced by approximately 8-fold. The mutant enzyme exhibits normal thermal stability but drastically altered reactivity toward p-hydroxymercuribenzoate. The catalytic subunit of the mutant and wild-type enzymes have very similar properties. These results, in conjunction with previous experiments, suggest that the intersubunit link involving Asp-236 is involved in the stabilization of the 240s loop in its tight-state position and that the regulatory subunits exert their effect on the catalytic subunits by influencing the position of the 240s loop.