Mechanism-Based Inactivation of VanX, a D-ALANYL-D-alanine Dipeptidase Necessary for Vancomycin Resistance
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- 作者：Romulo Ará ; oz ; Elizabeth Anhalt ; Loï ; c René ; Marie-Ange Badet-Denisot ; Patrice Courvalin ; and Bernard Badet
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：December 26, 2000
- 年：2000
- 卷：39
- 期：51
- 页码：15971 - 15979
- 全文大小：101K
- 年卷期：v.39,no.51(December 26, 2000)
- ISSN：1520-4995
文摘
VanX is a zinc-dependent <FONT SIZE="-1">D-Ala-D-Ala amino dipeptidase required for high-level resistanceto vancomycin. The enzyme is also able to process dipeptides with bulky C-terminal amino acids [Wu,Z., Wright, G. D., and Walsh, C. T. (1995) Biochemistry 34, 2455-2463]. We took advantage of thisobservation to design and synthesize the dipeptide-like D-Ala-D-Gly(S
fchars/Phi.gif" BORDER=0 >p-CHF2)-OH (7) as a potentialmechanism-based inhibitor. VanX-mediated peptide cleavage generates a highly reactive 4-thioquinonefluoromethide which is able to covalently react with enzyme nucleophilic residues, resulting in irreversibleinhibition. Inhibition of VanX by 7 was time-dependent (Kirr = 30 ± 1 
f">M; kinact = 7.3 ± 0.3 min-1) andactive site-directed, as deduced from substrate protection experiments. Nucleophilic compounds such assodium azide, potassium cyanide, and glutathione did not protect the enzyme from inhibition, indicatingthat the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivatethe dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction thatleads to inactivation. Inactivation was associated with the elimination of fluoride ion as deduced from 19FNMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data areconsistent with suicide inactivation of VanX by dipeptide 7. The small size of the VanX active site andthe presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D.E., et al. (1998) Mol. Cell 2, 75-84] associated with the high observed partition ratio of 7500 ± 500suggest that the inhibitor is likely to react at the entrance of the active site cavity.
fchars/Phi.gif" BORDER=0 >p-CHF2)-OH (7) as a potentialmechanism-based inhibitor. VanX-mediated peptide cleavage generates a highly reactive 4-thioquinonefluoromethide which is able to covalently react with enzyme nucleophilic residues, resulting in irreversibleinhibition. Inhibition of VanX by 7 was time-dependent (Kirr = 30 ± 1 f">M; kinact = 7.3 ± 0.3 min-1) andactive site-directed, as deduced from substrate protection experiments. Nucleophilic compounds such assodium azide, potassium cyanide, and glutathione did not protect the enzyme from inhibition, indicatingthat the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivatethe dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction thatleads to inactivation. Inactivation was associated with the elimination of fluoride ion as deduced from 19FNMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data areconsistent with suicide inactivation of VanX by dipeptide 7. The small size of the VanX active site andthe presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D.E., et al. (1998) Mol. Cell 2, 75-84] associated with the high observed partition ratio of 7500 ± 500suggest that the inhibitor is likely to react at the entrance of the active site cavity.