Mechanism for acivicin inactivation of triad glutamine amidotransferases

被引:78
作者
Chittur, SV [1 ]
Klem, TJ [1 ]
Shafer, CM [1 ]
Davisson, VJ [1 ]
机构
[1] Purdue Univ, Dept Med Chem & Mol Pharmacol, W Lafayette, IN 47907 USA
关键词
D O I
10.1021/bi0014047
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Eschcerichia 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 greater than or equal to 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,SR) 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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页码:876 / 887
页数:12
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