Site-directed mutagenesis of the regulatory domain of Escherichia coli carbamoyl phosphate synthetase identifies crucial residues for allosteric regulation and for transduction of the regulatory signals

Carbamoyl phosphate (CP), the essential precursor of pyrimidines and argini ne, is made in Escherichia coli by a single carbamoyl phosphate synthetase (CPS) consisting of 41.4 and 117.7 kDa subunits, which is feed-back inhibit ed by UMP and activated by IMP and ornithine. The large subunit catalyzes C P synthesis from ammonia in three steps, and binds the effecters in its 15 kDa C-terminal domain. Fifteen site-directed mutations were introduced in 1 3 residues of this domain to investigate the mechanism of allosteric modula tion by UMP and IMP. Two mutations, K993A and V994A, decreased significantl y or abolished enzyme activity, apparently by interfering with the step of carbamate synthesis, and one mutation, T974A, negatively affected ornithine activation. S948A, K954A, T974A, K993A and K993W/H995A abolished or greatl y hampered IMP activation and UMP inhibition as well as the binding of both effecters, monitored using photoaffinity labeling and ultracentrifugation binding assays. V994A also decreased significantly IMP and UMP binding. L99 0A, V991A, H995A, G997A and G1008A had more modest effects or affected more the modulation by and the binding of one than of the other nucleotide. K99 3W, R1020A, R1021A and K1061A were without substantial effects. The results confirm the independence of the regulatory and catalytic centers, and also confirm functional predictions based on the X-ray structure of an IMP-CPS complex. They prove that the inhibitor UMP and the activator IMP bind in th e same site, and exclude that the previously observed binding of ornithine and glutamine in this site were relevant for enzyme activation. K993 and V9 94 appear to be involved in the transmission of the regulatory signals trig gered by UMP and TMP binding. These effecters possibly change the position of K993 and V994, and alter the intermolecular contacts mediated by the reg ulatory domain. (C) 2000 Academic Press.