title: Inactivation of mitochondrial carbamoyl phosphate synthetase induced by ascorbate, oxygen, and Fe3+ in the presence of acetylglutamate: protection by ATP and HCO3- and lack of inactivation of ornithine transcarbamylase.
abstract: Of the two mitochondrial enzymes of the urea cycle, carbamoyl phosphate synthetase (CPS) was and ornithine transcarbamylase (OTC) was not inactivated by the Fe3+-oxygen-ascorbate model system for mixed-function oxidation [R. L. Levine, (1983) J. Biol. Chem. 258, 11828-11833]. The susceptibility of OTC was not increased by its substrates, products, or inhibitors, whereas that of CPS was markedly increased by acetylglutamate (its allosteric activator) when ATP was absent. Thus, acetylglutamate binds in the absence of ATP and exposes to oxidation essential groups of the enzyme. We estimate for this binding a KD value of 1.6 mM, which greatly exceeds the KD values (less than 10 microM) determined in the presence of ATP and bicarbonate. ATP, and even more, mixtures of ATP and bicarbonate protected CPS from inactivation. Acetylglutamate exposes the site for the ATP molecule that yields Pi, and it appears that ATP protects by binding at this site. Experiments of limited proteolysis with elastase suggest that oxidation prevents this binding of ATP and show that it accelerates cleavage of CPS by the protease, thus supporting the idea that oxidation may precede proteolysis. Trypsin, chymotrypsin, and papain also hydrolyze the oxidized enzyme considerably faster than the native enzyme. Our results also support the idea that oxidative inactivation is site specific and requires sites on the enzyme for Me2+ and, possibly, for a nucleotide.
authors.affiliation: Laboratory of Cell Chemistry, Instituto de Investigaciones Citológicas de la Caja de Ahorros de Valencia, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Alonso
authors.forename: V
authors.initials: V
authors.lastname: Rubio
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D001639:Bicarbonates / Q000494:pharmacology*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005971:Glutamates / Q000494:pharmacology*; D007501:Iron / Q000494:pharmacology*; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors*; D008930:Mitochondria, Liver / Q000201:enzymology*; D009954:Ornithine Carbamoyltransferase / Q000378:metabolism*; D010100:Oxygen / Q000494:pharmacology*; D051381:Rats
chemical_list: D001639:Bicarbonates; D005971:Glutamates; D000255:Adenosine Triphosphate; D007501:Iron; D009954:Ornithine Carbamoyltransferase; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C016195:N-acetylglutamic acid; D001205:Ascorbic Acid; D010100:Oxygen