Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase in the 20-kilodalton carboxy-terminal domain

Abstract

Mammalian liver mitochondrial carbamoyl phosphate synthetase, a polypeptide of 160 kDa, is activated allosterically by N-acetyl-L-glutamate. The analogue of this activator N-(chloroacetyl)-L-[14C]glutamate has been found to serve as a photoaffinity label for this enzyme. The specificity was demonstrated by the drastic reduction in the radioactivity bound to the protein when (a) an excess of unlabeled acetylglutamate was present during the irradiation and (b) the enzyme was replaced by pyruvate kinase, an enzyme that is not affected by acetylglutamate. The labeling was due to the photoactivation of the chloroacetyl group since there was no labeling under equal conditions with acetyl[14C]glutamate. To localize the binding site, limited proteolysis was used. Trypsin cleaves carbamoyl phosphate synthetase into complementary NH2- and COOH-terminal fragments of about 140 and 20 kDa, respectively [Powers-Lee, S. G., & Corina, K. (1986) J. Biol. Chem. 261, 15349-15352], but only the latter was found to be labeled. Similarly, of the various fragments generated by elastase, only two, of 20 and 120 kDa, contain the COOH terminus [see Powers-Lee and Corina (1986) above] and were found to be labeled. Thus, the binding site for acetylglutamate is within 20 kDa from the COOH terminus. This excludes the possibility that the acetylglutamate binding site evolved from an ancestral substrate site for glutamine: this substrate binds to the small subunit of the Escherichia coli enzyme, which is homologous to the NH2-terminal domain of the rat liver enzyme. Exhaustive tryptic digestion of photolabeled carbamoyl phosphate synthetase yielded a single radioactive peak, suggesting that the labeling is restricted to a single minimal tryptic peptide.

Role of Cys-1327 and Cys-1337 in Redox Sensitivity and Allosteric Monitoring in Human Carbamoyl Phosphate Synthetase

“…The mutations appeared to additionally affect interaction with AGA, with mutant apparent K m values that were 7-10-fold lower than for wild type hCPS. This observation was consistent with the previous reports that the C-terminal region of CPS is involved in binding AGA (24,25). Interaction with ammonia was not significantly affected by the mutations.…”

Required Allosteric Effector Site for -Acetylglutamate on Carbamoyl-Phosphate Synthetase I

“…In contrast to the CPSase II effectors, AGA greatly facilitates the binding of ATP B to CPS I but has no detectable effect on the binding of ATP C (22,30). In the single previous study of AGA site localization, reaction of rat liver CPSase I with the photoaffinity label N-chloroacetyl[ 14 C]glutamate was found to result in labeling of only the C-terminal region of CPSase (31).…”

Regulation of the mammalian carbamoyl‐phosphate synthetase II by effectors and phosphorylation

“…We have preliminary evidence that UTP binds to the C-terminal domain of the CPS I1 region in CAD (Shaw, Sultan and Carrey, unpublished results). The analogous region has been implicated in binding the allosteric activator N-acetylglutamate to the mammalian CPS I enzyme [7,441 and UMP (inhibitor) to the E. coli enzyme [8]. In CPS I the C-terminal region has been implicated in binding ATP [41,421, but there seems to be no direct involvement in ATP-binding in CAD [40] and so UTP may stabilise the less active form of CPS I1 by indirect conformational changes.…”

“…This site is found C-terminal to the duplicated ATPbinding domains in CAD [6]. In other CPS enzymes a Cterminal domain is implicated in binding effectors [7,81. If the same region in CAD perfoms both of these functions, then reversible phosphorylation and the binding of effectors could act through similar conformational mechanisms to regulate the biosynthesis of carbamoyl phosphate by CPS I1 in CAD.…”