Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain.
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.
Rodriguez-Aparicio
L B
LB
Department of Cell Biochemistry, Instituto de Investigaciones Citológicas de la Caja de Ahorros de Valencia (Centro Asociado del CSIC), Spain.
Guadalajara
A M
AM
Rubio
V
V
D000345:Affinity Labels / Q000378:metabolism*; D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010449:Peptide Mapping; D051381:Rats; D014357:Trypsin
D000345:Affinity Labels; D005971:Glutamates; C060263:N-(chloroacetyl)glutamate; D014357:Trypsin; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C016195:N-acetylglutamic acid