Abstract
Escherichia coli aspartate transcarbamoylase regulates pyrimidine biosynthesis by altering its activity homotropically in response to one of its substrates and heterotropically in response to nucleotide effectors. The mechanism of this regulation involves two structurally and functionally different forms of the enzyme, one with low activity and low affinity for substrates (T state) and the other with high activity and high affinity for substrates (R state). Heterotropic regulation may be due to the direct transmission of a regulatory "signal" between the regulatory site and the active site some 60 A away and/or may involve altering the relative stability of the two forms of the enzyme. By combining a T state-stabilized mutant version of the enzyme, previously thought to have a defect in a heterotropic transmission pathway, with a known R state-stabilized mutant enzyme, we were able to restore some properties of the wild-type enzyme. These data imply that the relative stabilization of the T and R states of the enzyme is in part responsible for the homotropic and heterotropic properties of aspartate transcarbamoylase and that direct pathways for transmission of the heterotropic signals are unlikely.
Highlights
Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) catalyzes the committed step in the biosynthesis of pyrimidine nucleotides: the reaction between carbamyl phosphate and Laspartate to form N-carbamyl-L-aspartate and Pi [1, 2]
These data imply that the relative stabilization of the T and R states of the enzyme is in part responsible for the homotropic and heterotropic properties of aspartate transcarbamoylase and that direct pathways for transmission of the heterotropic signals are unlikely
One key unanswered question, pertaining to the entire class of allosteric enzymes, is the molecular mechanism by which these enzymes alter their activity in response to the binding of regulatory molecules at site(s) remote from the active site; it is this question that we address with the experiments described here
Summary
(Received for publication, June 13, 1996, and in revised form, September 11, 1996). Jennifer M. The extent of the ATP inhibition and the strength of its interaction with the enzyme are the same as that observed with CTP [17] These studies, involving mutants at the interface between the zinc and allosteric domains of the regulatory chains, have led to the proposal that the influence of the heterotropic effectors is transmitted from the regulatory binding site to the active site via a specific pathway involving helix H2Ј of the regulatory chain [16]. Another possible interpretation of the effects of these mutations is that they alter the global stabilization of either the T or R states of the enzyme. Such a complementation would suggest that direct pathways for transmission of the heterotropic signals are unlikely
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