Mechanism-based inactivation of VanX, a D-alanyl-D-alanine dipeptidase necessary for vancomycin resistance

VanX is a zinc-dependent D-Ala-D-Ala amino dipeptidase required for high-le vel resistance to 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 this observation to design and synthesize the dipeptide-like D-Ala-D-Gly(S Phip-CHF2)-OH (7) as a potential mechanism-based inhibitor. VanX-mediated peptide cleavage g enerates a highly reactive 4-thioquinone fluoromethide which is able to cov alently react with enzyme nucleophilic residues, resulting in irreversible inhibition. Inhibition of VanX by 7 was time-dependent (K-irr = 30 +/- 1 mu M; k(inact) = 7.3 +/- 0.3 min(-1)) and active site-directed, as deduced fro m substrate protection experiments. Nucleophilic compounds such as sodium a zide, potassium cyanide, and glutathione did not protect the enzyme from in hibition, indicating that the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivate the dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction that leads to inactivation. Inactivation was associated with the eliminati on of fluoride ion as deduced from F-19 NMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data are consisten t with suicide inactivation of VanX by dipeptide 7. The small size of the V anX active site and the presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D. E., et al. (1998) Mel. Cell 2, 75-84] associated with the high observed partition ratio of 7500 +/ - 500 suggest that the inhibitor is likely to react at the entrance of the active site cavity.