Catalytic-regulatory subunit interactions and allosteric effects in aspartate transcarbamylase.
Academic Article
Overview
abstract
The x-ray structure of the unliganded aspartate transcarbamylase reveals that Arg-113 of the catalytic chain is involved in an important set of interactions at the interface between the catalytic and regulatory subunits (Honzatko, R.B., Crawford, J.L., Monaco, H.L., Ladner, J.E., Edwards, B.F.P., Evans, D.R., Warren, S.G., Wiley, D.C., Ladner, R.C., and Lipscomb, W. N. (1982) J. Mol. Biol. 160, 219-263). In order to disturb this interaction, site-directed mutagenesis has been used to replace Arg-113 with glycine. This modification results in a substantial weakening of the interface between the catalytic and regulatory subunits leading to a high tendency for dissociation. The unliganded mutant enzyme exhibits a pH dependence and a sensitivity toward mercurials analogous to that obtained for the relaxed conformation of the wild-type enzyme. Moreover, the presence of saturating concentrations of aspartate is accompanied by only a slight shift in the optimal pH for activity. The bisubstrate analog N-(phosphonacetyl)-L-aspartate induces a 2-fold increase in the sulfhydryl reactivity as compared to the 4-fold increase observed for the wild-type enzyme. Despite this change in the interactions at the interface between the catalytic and regulatory subunits, the mutant enzyme still retains homotropic and heterotropic effects and exhibits a normal affinity for aspartate. Together these data show that a substantial weakening of the catalytic-regulatory interface can occur without altering the allosteric properties of the enzyme. These results also indicate that the intersubunit interactions involving Arg-113, between the polar domain of the catalytic chain and the zinc domain of the regulatory chain, do not participate in the homotropic cooperativity of the enzyme.
