Regulation of mitochondrial carbamoyl-phosphate synthetase 1 activity by active site fatty acylation.
In addition to its role in reversible membrane localization of signal-transducing proteins, protein fatty acylation could play a role in the regulation of mitochondrial metabolism. Previous studies have shown that several acylated proteins exist in mitochondria isolated from COS-7 cells and rat liver. Here, a prominent fatty-acylated 165-kDa protein from rat liver mitochondria was identified as carbamoyl-phosphate synthetase 1 (CPS 1). Covalently attached palmitate was linked to CPS 1 via a thioester bond resulting in an inhibition of CPS 1 activity at physiological concentrations of palmitoyl-CoA. This inhibition corresponds to irreversible inactivation of CPS 1 and occurred in a time- and concentration-dependent manner. Fatty acylation of CPS 1 was prevented by preincubation with N-ethylmaleimide and 5'-p-fluorosulfonylbenzoyladenosine, an ATP analog that reacts with CPS 1 active site cysteine residues. Our results suggest that fatty acylation of CPS 1 is specific for long-chain fatty acyl-CoA and very likely occurs on at least one of the essential cysteine residues inhibiting the catalytic activity of CPS 1. Inhibition of CPS 1 by long-chain fatty acyl-CoAs could reduce amino acid degradation and urea secretion, thereby contributing to nitrogen sparing during starvation.
Corvi
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MM
Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Soltys
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Berthiaume
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LG
D000215:Acylation; D000818:Animals; D001665:Binding Sites; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D005033:Ethylmaleimide / Q000494:pharmacology; D005227:Fatty Acids / Q000378:metabolism*; D019811:Hydroxylamine / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013367:Submitochondrial Particles / Q000201:enzymology / Q000378:metabolism; D013379:Substrate Specificity
D005227:Fatty Acids; D010171:Palmitoyl Coenzyme A; D019811:Hydroxylamine; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D005033:Ethylmaleimide