VanX protein is a D‐alanyl‐D‐alanine dipeptidase essential for vancomycin resistance in Enterococcus. It is also a key drug target in circumventing clinical glycopeptide antibiotic resistance. The dipeptide‐like compound D‐Ala‐D‐Gly(SC6H4‐p‐CHF2) (1) was recently reported as the first mechanism‐based inhibitor with high affinity for the enzyme but poor inhibitory efficiency (kinact/Kirr = 9320 M−1 s−1) and a high partition ratio (7600). In order to assess the effects of variations in aromatic substituents on the in vitro inhibition properties of 1, we have prepared a few derivatives and analyzed them using the alternative substrate D‐Ala‐D‐Gly(SPh)‐OH (15) designed according to a similar strategy. Whereas moving the electrophilic difluoromethyl group to the ortho position slightly improved the inhibition parameters (kinact/Kirr = 1140 M−1 s−1) with a small decrease in the partition ratio (7200), introduction of a methoxy group in D‐Ala‐D‐ortho/para‐(difluoromethyl)phenylthioglycines resulted in a marked decrease in inhibitory efficiency. These results demonstrate the difficulties in improving the leading compound 1 given the restricted space available at the VanX active site. (© Wiley‐VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Oligomerization can endow proteins with novel structural and catalytic properties. The native dimer of bovine seminal ribonucleases (BS-RNase) binds, melts and catalyses the hydrolysis of double-stranded ribonucleic acids 30-fold better than its pancreatic homologue, the monomeric RNase A. Chemically induced oligomers of pancreatic RNase A are also found to show an increase in enzyme activity on double-stranded poly(A).poly(U) (Libonati, M. Bertoldi, M. and Sorrentino, S. (1996) Biochem. J. 318, 287-290) and, therefore, can be considered as potential immunosuppressive and cytotoxic agents. We report here a study on the relationship between surface histidine topography in oligomeric forms of these ribonucleases and their catalytic properties. Subtle changes in structure conformation of both BS-RNase and oligomeric RNase A are shown to result in a modification of the affinity of these proteins toward the immobilized transition-metal chelate, IDA-Cu(II). Because, such conformational change has been shown to correlate with an improvement of the newly acquired biological activities upon oligomerization, we can conclude that surface histidines topography constitutes an exquisite probe for the study of protein structure/function relationship.

A direct continuous UV-Vis spectrophotometric assay has been developed for VanX, a D-alanyl-D-alanine aminodipeptidase necessary for vancomycin resistance. This method is based on the hydrolysis of the alternative substrate D-alanyl-alpha-(R)-phenylthio-glycine D-Ala-D-Gly(S-Ph)-OH (H-DAla-DPsg-OH, 5a). Spontaneous decomposition of the released phenylthioglycine generates thiophenol, which is quantified using Ellman's reagent. The dipeptide behaved as an excellent substrate of VanX, exhibiting Michaelis-Menten kinetics with a kcat of 76 +/- 5/s and a KM of 0.83 +/- 0.08 mm (kcat = 46 +/- 3/s and KM = 0.11 +/- 0.01 mm for D-Ala-D-Ala). Determination of the kinetic parameters of the previously reported mechanism-based inhibitor D-Ala-D-Gly(SPhip-CHF2)-OH (H-D-Ala-DPfg-OH, 5c) [Araoz, R., Anhalt, E., René, L., Badet-Denisot, M.-A., Courvalin, P. & Badet, B. (2000) Biochemistry 39, 15971-15979] using the substrate reported in the present study yielded values of Kirr of 22 +/- 1 microM and kinact of 9.3 +/- 0.4/min in good agreement with values previously obtained in our laboratory (Kirr = 30 +/- 1 mm; kinact = 7.3 +/- 0.3/min). In addition, inhibition by the competing substrate D-Ala-D-Ala resulted in determination of a Ki = 70 +/- 6 microM close to the previously reported KM value. These results demonstrate that the present assay is a convenient, rapid and sensitive tool in the search for VanX inhibitors.