Mechanism for acivicin inactivation of triad glutamine amidotransferases

Acivicin [(alphaS,5S)-alpha -amino-3-chloro-4,5-dihydro-5-isoxazoleacetic a cid] was investigated as an inhibitor of the triad glutamine amidotransfera ses, IGP synthase and GMP synthetase, Nucleophilic substitution of the chlo rine 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 modifica tion in the heterodimeric IGPS from Eschcerichia coli (HisHF) by tryptic di gest and FABMS, Distinctions in the glutaminase domains of IGPS from E, col i, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. co li GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-depend ent reaction was inhibited with unit stoichiometry, In analogy to the condi tional glutaminase activity seen in IGPS and GMPS, the rates of inactivatio n were accelerated greater than or equal to 25-fold when a nucleotide subst rate (or analogue) was present. The specificity (k(inact)/K-i(app)) for aci vicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested u nder identical conditions as acivicin and showed little inhibitory effect o n the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a gl utamine amidotransferase mechanism-based covalent bond formation in the pre sence of nucleotide substrates or products. Acivicin and its (alphaS,SR) di astereomer were modeled in the glutaminase active site of GMPS and CPS to c onfirm that the binding orientation of the dihydroisoxazole ring is identic al in all three triad glutamine amidotransferases, Stabilization of the imi ne-thioether intermediate by the oxyanion hole in triad glutamine amidotran sferases appears to confer the high degree of specificity for acivicin inhi bition and relates to a common mechanism for inactivation.