Relationship between domain closure and binding, catalysis, and regulation in Escherichia coli aspartate transcarbamylase

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Abstract
Previous evidence, from both crystallographic and biochemical studies, has indicated that profound tertiary and quaternary changes in the structure of Escherichia coli aspartate transcarbamylase occur upon the binding of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate (PALA). In particular, within a single catalytic polypeptide chain, the aspartate binding domain relocates closer to the carbamyl phosphate binding domain, thereby resulting in a major reorganization of the interface between the two domains. Among the new interactions, salt bridges between Glu-50 and both Arg-167 and Arg-234 are formed. In the present study, site-directed mutagenesis is used to replace Glu-50 by glutamine in the catalytic chain. The Michaelis constant for aspartate of the mutant catalytic subunit is about 10-fold higher and the turnover number 10-fold lower than their respective counterparts in the wild-type catalytic subunit, whereas the dissociation constant for carbamyl phosphate is almost unchanged. For the holoenzyme, this substitution results in an 8-fold decrease in the specific activity, a 20-fold increase in the aspartate concentration that gives half of the maximal velocity, and a loss of cooperativity for both substrates. However, the mutant enzyme is not "frozen" in a low-affinity-low-activity conformation since PALA stimulates the activity severalfold and induces an increase in the sulfhydryl reactivity analogous to that of the wild-type enzyme. Together these results indicate that the stabilization of the aspartate binding domain near the carbamyl phosphate binding domain, through specific interdomain bridging interactions, is necessary for the high-affinity-high-activity configuration of the active site. The importance of these salt bridges for the homotropic and heterotropic interactions in aspartate transcarbamylase is discussed.