Oxidative inactivation of carbamoyl phosphate synthetase (ammonia). Mechanism and sites of oxidation, degradation of the oxidized enzyme, and inactivation by glycerol, EDTA, and thiol protecting agents.
Acetylglutamate and ATP accelerate the oxidative inactivation of carbamoyl phosphate synthetase I by mixtures of Fe3+, ascorbate, and O2, but the mechanism of the inactivation differs with each ligand. In the presence of acetylglutamate, MgATP prevents, Mg2+, Mn2+, and catalase have no effect, and EDTA increases the inactivation, and the two phosphorylation steps of the enzyme reaction are lost simultaneously. The inactivation appears to be mediated by dehydroascorbate and is associated with the reversible oxidation of the highly reactive cysteines 1327 and 1337 and with oxidation of non-thiolic groups in the second 40-kDa domain (the enzyme consists of 4 domains of 40, 40, 60, and 20 kDa, from the amino terminus). The data are consistent with oxidation of groups at or near the site for ATPA (ATPA yields Pi; ATPB yields carbamoyl phosphate), and with the location of this site at the interphase between the second 40-kDa and the COOH-terminal domains. The oxidative inactivation promoted by ATP is inhibited by Mg2+, Mn2+, catalase, and EDTA, is not mediated by dehydroascorbate, and is not associated with oxidation of cysteines 1327 and 1337. Groups in the 60-kDa domain are oxidized. The phosphorylation step involving ATPB is lost preferentially, and the inactivation and the binding of ATPB exhibit the same dependency on the concentration of ATP. The results indicate that the oxidation is catalyzed by FeATP bound at the site for ATPB and support the binding of ATPB in the 60-kDa domain. We also demonstrate that mercaptoethanol, reducing impurities in glycerol, and dithioerythritol, in the presence of EDTA, replace ascorbate in the oxidative system. In addition, we study the influence of the oxidation on the degradation of the enzyme by rat liver lysosomes, mitochondria, and cytosol.
Alonso
E
E
Instituto de Investigaciones Citológicas, F. I. B. (Centro Asociado del CSIC), Valencia, Spain.
Cervera
J
J
García-España
A
A
Bendala
E
E
Rubio
V
V
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002413:Cations, Divalent; D004226:Dithioerythritol / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000494:pharmacology*; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology; D010084:Oxidation-Reduction; D051381:Rats
D002413:Cations, Divalent; D008345:Manganese; D004226:Dithioerythritol; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D004492:Edetic Acid; D002374:Catalase; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D008274:Magnesium; D005990:Glycerol