Lysine-60 in the regulatory chain of aspartate transcarbamoylase has been changed to an alanine by site-specific mutagenesis. The resulting enzyme exhibits activity and homotropic cooperativity identical with those of the wild-type enzyme. The substrate concentration at half the maximal observed specific activity decreases from 13.3 mM for the wild-type enzyme to 9.6 mM for the mutant enzyme. ATP activates the mutant enzyme to the same extent that it does the wild-type enzyme, but the concentration of ATP required to reach half of the maximal activation is reduced approximately 5-fold for the mutant enzyme. CTP at a concentration of 10 mM does not inhibit the mutant enzyme, while under the same conditions CTP at concentrations less than 1 mM will inhibit the wild-type enzyme to the maximal extent. Higher concentrations of CTP result in some inhibition of the mutant enzyme that may be due either to hetertropic effects at the regulatory site or to competitive binding at the active site. UTP alone or in the presence of CTP has no effect on the mutant enzyme. Kinetic competition experiments indicate that CTP is still able to displace ATP from the regulatory sites of the mutant enzyme. Binding measurements by equilibrium dialysis were used to estimate a lower limit on the dissociation constant for CTP binding to the mutant enzyme (greater than 1 x 10(-3) M). Equilibrium competition binding experiments between ATP and CTP verified that CTP still can bind to the regulatory site of the enzyme. For the mutant enzyme, CTP affinity is reduced approximately 100-fold, while ATP affinity is increased by 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)