Cytidine triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the K m and IC 50 values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. 568 is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the V max of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser 568 as a major site of CTPS2 regulation by phosphorylation.Cytidine nucleotides are precursors for the synthesis of nucleic acids and phospholipids (1-4) as well as the post-translational modification of proteins by sialylation (1-3) and the regulation of enzymes such as CTP-phosphocholine cytidylyltransferase (2), carbamoyl phosphate synthetase, and phosphatidylserine synthase (5). CTP is the nucleotide with the lowest cellular concentration (1, 6), making it rate-limiting for RNA synthesis and other CTP-dependent events. Hence, understanding the regulation of CTP synthesis is important for many growth-related processes.CTP synthetase (CTPS, EC 6.3.4.2) 4 is the rate-limiting enzyme for the synthesis of cytosine nucleotides from both the de novo and uridine salvage pathways (7). CTPS consists of two enzymatic domains. In the synthase domain, UTP is phosphorylated by the ␥-phosphoryl group of ATP to form 4-phospho-UTP via an ATPase reaction (8 -11). In the glutaminase domain, the substrate glutamine is hydrolyzed to glutamate and NH 3 (8), and the resultant NH 3 is then transported to the synthase domain active site through an NH 3 tunnel (11, 12). NH 3 is then transferred to the phosphorylated UTP to form CTP (8). GTP activates the reaction by accelerating the formation of a covalent glutamyl-enzyme catalytic intermediate (8). CTPS has three functionally distinct sites: the glutaminase site for glutamine hydrolysis, the active site where CTP is formed, and the allosteric site where GTP binds (13). CTPS is also regulated by feedback inhibition, allowing the product CTP to inhibit the activity of the enzyme (14 -17).There are two isoforms of CTPS in humans, CTPS1 and CTPS2, and they share 74% amino acid identity (...

No abstract

Mass spectrometric methods of determining protein ubiquitination are described. Characteristic mass shifts and fragment ions indicating ubiquitinated lysine residues in tryptic and gluC digests are discussed. When a ubiquitinated protein is enzymatically digested, a portion of the ubiquitin side chain remains attached to the modified lysine. The ubiquitinated peptide thus has two N-termini - one from the original peptide and one from the ubiquitin side chain. Thus, it is possible to have two series of b ions and y ions, the additional series is the one that includes fragments containing portions of the ubiquitin side chain. Any diagnostic ions for the modification must include portions of this side chain. Fragment ions involving any part of the "normal" peptide will vary in mass according to the peptide being modified and will therefore not be of general diagnostic use. These diagnostic ions, found through examination of the MS/MS spectra of model ubiquitinated tryptic and gluC peptides, have not previously been reported. These ions can be used to trigger precursor ion scanning in automated MS/MS data acquisition scanning modes.

No abstract

Cytidine triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the K m and IC 50 values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. Metabolic labeling of hCTPS2 with [ 32 P]H 3 PO 4 demonstrated that Ser 568 and Ser 571 were two major phosphorylation sites, and additional studies demonstrated that Ser 568 was phosphorylated by casein kinase 1 both in vitro and in vivo. Interestingly, mutation of Ser 568 (S568A) but not Ser 571 significantly increased hCTPS2 activity, demonstrating that Ser 568 is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the V max of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser 568 as a major site of CTPS2 regulation by phosphorylation.