Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.3.5.5 (CPS)
 1,262 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Acivicin [L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid; NSC 163501] is a fermentation-derived amino acid antibiotic antagonistic to L-glutamine which exhibits potent oncolytic properties. We have developed a variant of P388 leukemia resistant to acivicin (P388/ACIA) and compared its properties with those of the parent line (P388/S). An examination of the enzymes utilizing L-glutamine revealed that the basal specific activities of L-asparagine synthetase and L-glutaminase were 1-to 3-fold higher in the parent line. The activities of carbamoyl phosphate synthetase II, L-asparagine synthetase, formylglycinamide ribonucleotide amidotransferase, and guanosine monophosphate synthetase were about equally inhibited in the two cell lines, while there was a partial inhibition of 5-phosphoribosyl-1-pyrophosphate amidotransferase, fructose-6-phosphate amidotransferase, and L-glutaminase activities, found only in the sensitive line. Cytidine triphosphate synthetase activity was not inhibited in either line. There was no difference in the dose response or restitution of L-glutamine utilizing enzyme activities between the two lines. Acivicin treatment produced a 2- to 3-fold augmentation of the L-glutamine pools only in the sensitive line. Drug injection induced increased 5-phosphoribosyl-1-pyrophosphate levels in both lines. Acivicin perturbed guanosine nucleotide pools only in the sensitive line, indicating that the primary mechanism of action of acivicin in P388 leukemia may be directed at guanosine monophosphate synthetase. Transport studies demonstrated a restricted uptake of acivicin by the resistant cells. These studies suggest that the transport of acivicin and L-glutamine plays an important role in determining the sensitivity or resistance to acivicin in these tumors.
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PMID:Mechanism of resistance of a variant of P388 leukemia to L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin). 257 92

The in vivo actions of two antimetabolites, acivicin (NSC-163501) and tiazofurin (NSC-286193), were examined on the enzymic programs of rat bone marrow. From the bone marrow of the femurs, 100,000 g supernatant fractions were prepared; enzymic activities were measured by isotopic assays, and cellularity was determined. In the normal bone marrow, the specific activities of pyrimidine de novo synthetic enzymes, CDP reductase, dTMP synthase, CTP synthase, carbamoyl-phosphate synthase II (synthase II), orotidine 5'-phosphate decarboxylase and aspartate carbamoyltransferase, were 1, 2.7, 5, 10, 63 and 601 nmol/hr/mg protein, respectively, whereas those of the salvage enzymes, deoxycytidine, thymidine, cytidine and uridine kinases were 3, 43, 149, and 367 nmol/hr/mg protein, respectively. In purine biosynthesis, the activities of the de novo synthetic enzymes, IMP dehydrogenase, formylglycinamidine ribonucleotide (FGAM) synthase, GMP synthase, amidophosphoribosyl-transferase (AT) and adenylosuccinate synthase were 16, 8, 107, 78 and 124 nmol/hr/mg protein, respectively, and those of the salvage enzymes, adenine, hypoxanthine and guanine phosphoribosyl-transferases, were 340, 407, and 1018 nmol/hr/mg protein, respectively. The sequence of events was elucidated after a single i.p. injection of acivicin (5 mg/kg) or tiazofurin (200 mg/kg). Within 2 hr after acivicin injection, CTP, GMP and FGAM synthases lost 85-90%, while AT and synthase II lost 50 and 80%, respectively, of their activities. The activities rose to near normal range by 72-96 hr. The bone marrow cellularity decreased, reaching a nadir at 24 and 48 hr, and returning to normal range by 72 and 92 hr; thymidine kinase activity followed a similar pattern. Tiazofurin injection depressed IMP dehydrogenase activity to 20% by 2 hr with a rebound to normal range by 48 and 72 hr. The cellularity decreased more slowly, reaching its lowest point at 24 hr and returning to normal range at 72 hr. For acivicin the marked depletion of the activities of the glutamine-utilizing enzymes and for tiazofurin that of IMP dehydrogenase might account, in part at least, for the bone marrow toxicity of these antimetabolites. Because of the presence in the bone marrow of high activities of purine and pyrimidine salvage enzymes, it should be possible to design methods utilizing nucleosides and nucleobases to protect the bone marrow from the action of antimetabolites.
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PMID:Enzymic programs of rat bone marrow and the impact of acivicin and tiazofurin. 334

In contrast to several other glutamine amidotransferases including asparagine synthetase, cytidine 5'-triphosphate (CTP) synthetase, carbamoyl phosphate synthetase, and phosphoribosyl pyrophosphate (PRPP) amidotransferase, guanosine monophosphate synthetase (GMPS) will not utilize hydroxylamine as an alternative nitrogen source. Instead, the enzyme is inhibited by an unknown mechanism. One untested hypothesis was that hydroxylamine serves as a substrate and intercepts a xanthosine 5'-monophosphate- (XMP-) adenylate intermediate in the enzyme active site. The nucleotide product of this substitution reaction would be N2-hydroxyguanosine 5'-monophosphate (N2-OH-GMP, 2). Here we describe the chemoenzymatic preparation of 2, via the nucleotide 2-fluoroinosine 5'-monophosphate (F-IMP, 5), and characterization of both these compounds as inhibitors of Escherichia coli GMPS. F-IMP was conceived as an electronic mimic of a reactive intermediate in the GMPS reaction but was found to bind weakly to the enzyme (IC50 > 2 mM). In contrast, N2-OH-GMP shows time-dependent inhibition and is competitive with respect to XMP (Ki = 92 nM), representing the first example of a compound that displays these kinetic properties with GMPS. The mechanism of inhibition is proposed to occur via formation of a ternary E.ATP.2 complex, followed by a rate-determining isomerization to a higher affinity complex that has a t1/2 =7.5 min. The contrast in inhibitory activity for 2-substituted purines with GMPS formulates a basis for future inhibitor design. In addition, these results complement recent structural studies of GMPS and implicate the formation of the XMP-adenylate intermediate inducing a probable conformational change that stimulates the hydrolysis of glutamine.
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PMID:N2-hydroxyguanosine 5'-monophosphate is a time-dependent inhibitor of Escherichia coli guanosine monophosphate synthetase. 989 Sep 11

Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.
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PMID:Mechanism for acivicin inactivation of triad glutamine amidotransferases. 1117 Apr 8

Carbamoyl phosphate synthetase II encodes the first enzymic step of de novo pyrimidine biosynthesis. Carbamoyl phosphate synthetase II is essential for Toxoplasma gondii replication and virulence. In this study, we characterised the primary structure of a 28kb gene encoding Toxoplasma gondii carbamoyl phosphate synthetase II. The carbamoyl phosphate synthetase II gene was interrupted by 36 introns. The predicted protein encoded by the 37 carbamoyl phosphate synthetase II exons was a 1,687 amino acid polypeptide with an N-terminal glutamine amidotransferase domain fused with C-terminal carbamoyl phosphate synthetase domains. This bifunctional organisation of carbamoyl phosphate synthetase II is unique, so far, to protozoan parasites from the phylum Apicomplexa (Plasmodium, Babesia, Toxoplasma) or zoomastigina (Trypanosoma, Leishmania). Apicomplexan parasites possessed the largest carbamoyl phosphate synthetase II enzymes due to insertions in the glutamine amidotransferase and carbamoyl phosphate synthetase domains that were not present in the corresponding gene segments from bacteria, plants, fungi and mammals. The C-terminal allosteric regulatory domain, the carbamoyl phosphate synthetase linker domain and the oligomerisation domain were also distinct from the corresponding domains in other species. The novel C-terminal regulatory domain may explain the lack of activation of Toxoplasma gondii carbamoyl phosphate synthetase II by the allosteric effector 5-phosphoribosyl 1-pyrophosphate. Toxoplasma gondii growth in vitro was markedly inhibited by the glutamine antagonist acivicin, an inhibitor of glutamine amidotransferase activity typically associated with carbamoyl phosphate synthetase II, guanosine monophosphate synthetase, or CTP synthetase.
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PMID:Organisation and sequence determination of glutamine-dependent carbamoyl phosphate synthetase II in Toxoplasma gondii. 1254 50