Studies on the Subunit Interactions in Aspartate Transcarbamylase

Abstract

<p>The effects of subunit interactions on the function of aspartate transcarbamylase from Escherichia coli have been investigated by the characterization of an intermediate formed during the reassembly of the native enzyme (c₆r₆) from its constituent catalytic (c₃) and regulatory (r₂) subunits. The intermediary complex was formed by mixing a low concentration of c₃ with excess r₂ and under these conditions was stabilized against reassociation to the native c₆r₆ structure. Sucrose density gradient analysis indicated that the complex sedimented at a position intermediate between the native enzyme and the catalytic subunit. These and other results indicated that the complex has the structure c₃r₆. Similar sedimentation experiments showed the reversibility of c₃r₆ formation and its conversion to c₆r₆ at low r₂ concentrations.</p> <p>While the native enzyme gives markedly sigmoid initial velocity kinetics with respect to the substrate aspartate the c₃r₆ complex gives a hyperbolic dependence similar to c₃ but with a much reduced Km. The high affinity of c₃r₆ for aspartate suggests that the complex may represent the relaxed state of aspartate transcarbamylase. Further evidence for this was provided from studies of the pH activity profiles of the various enzyme forms.</p> <p>Although both regulatory and catalytic subunits are present in the c₃r₆ complex the allosteric inhibitor CTP and activator ATP were found to have no effect on c₃r₆.</p>