Abstract
Ornithine carbamoyltransferase (OTC) is a key enzyme in the urea cycle to detoxify ammonium produced from amino acid catabolism. OTC deficiency is an X-linked genetic disorder ranging from fatal in newborns to hyperammonemia and anorexia in adults. Through affinity purification of acetylated peptides and mass spectrometry, we identified that OTC is acetylated on lysine residues, including Lys88, which is also mutated in OTC-deficient patients. OTC acetylation was confirmed to occur under physiological conditions. Biochemical characterizations revealed that OTC Lys88 acetylation decreases the affinity for carbamoyl phosphate, one of the two OTC substrates, and the maximum velocity, whereas the K(m) for ornithine, the other OTC substrate, is not affected. Furthermore, Lys88 acetylation is regulated by both extracellular glucose and amino acid availability, indicating that OTC activity may be regulated by cellular metabolic status. Our results provide an example of the novel mechanism of regulating metabolic enzyme activity through protein acetylation.
Highlights
Because there is no alternative way of urea synthesis, blockade in the urea cycle results in devastating health consequences
Ornithine carbamoyltransferase (OTC) Is Acetylated at Lys88—Most reported acetylation studies are with nuclear proteins, whereas few cytoplasmic protein acetylation studies have been documented
The subcellular fractions were digested with trypsin, and acetylated peptides were purified by anti-acetyllysine antibody followed by tandem liquid chromatography-tandem mass spectrometry analysis
Summary
Cell Culture and Transfection— HEK293T and Chang liver cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal calf serum (HyClone), 100 units/ml penicillin, and streptomycin (Invitrogen). Glucose/Amino Acid Treatment— Cells or transfected cells were kept in Dulbecco’s modified Eagle’s medium for 24 h before treatment. Cells were washed twice with phosphate-buffered saline and continued culture in Dulbecco’s modified Eagle’s medium (without glucose but with essential amino acids; Sigma, D5030) with the desired sup-. For anti-FLAG immunoprecipitation (IP), 500 l of cell lysate was incubated with anti-FLAG M2-agarose for 4 h at 4 °C; for OTC antibody IP, lysate was incubated with anti-OTC antibody (Aviva Systems Biology; 1:500) overnight and protein A/G beads were added, and incubation was continued for another 2 h. 5 l of FLAG peptide-eluted ectopic OTC-FLAG solution or 10 l of OTC antibody-immunoprecipitated beads was added to a solution containing ornithine and triethanolamine to a final volume of 675 l. Citrulline production was determined by adding 47 l of 3% 2,3-butanedionemonoxime, boiling in the dark for 15 min, and reading absorbance at 490 nm
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