DOMAIN-STRUCTURE OF THE LARGE SUBUNIT OF ESCHERICHIA-COLI CARBAMOYL PHOSPHATE SYNTHETASE - LOCATION OF THE BINDING-SITE FOR THE ALLOSTERIC INHIBITOR UMP IN THE COOH-TERMINAL DOMAIN

The large subunit of Escherichia coli carbamoyl phosphate synthetase (a polypeptide of 117.7 kDa that consists of two homologous halves) is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus (demonstrated by NH2-terminal sequencing). UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [C-14]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradiation with [C-14]UMP. The [C-14]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. In the presence of SDS, the labeled large subunit is cleaved by trypsin or by V8 staphylococcal protease at a site located 15 or 25 kDa, respectively, from the COOH terminus (shown by NH2-terminal sequencing), and only the 15- or 25-kDa fragments are labeled. Similarly, upon cleavage of the aspartyl-prolyl bonds of the [C-14]UMP-labeled enzyme with 70% formic acid, labeling was found only in the 18.5-kDa fragment that contains the COOH terminus of the subunit. Thus, UMP binds to the COOH-terminal domain. Since the binding sites for IMP and UMP overlap, most probably IMP also binds in this domain. The protection from proteolysis by ornithine suggests that ornithine binds in the same domain. Acetylglutamate (the allosteric activator of the ureotelic enzyme) binds to the homologous COOH-terminal domain of the rat liver enzyme [Rodriguez-Aparicio, L., et al. (1989) Biochemistry 28, 3070-3074]. Thus, the COOH-terminal domain appears to be the regulatory domain of the carbamoyl phosphate synthetases. To account for the effects of the allosteric effectors on the binding of ATP, we propose a scheme where the two halves of the large subunit form a pseudohomodimer by complementary isologous association, thus placing the NH2 half, which is involved in the binding of the molecule of ATP that yields P(i), close to the regulatory domain.