Continuous assay for VanX, the D-Alanyl-D-Alanine dipeptidase required for high-level vancomycin resistance

The reaction of L-alanine-p-nitroanilide with VanX was studied in an effort to develop a continuous assay for VanX activity for future kinetic and inhibition studies. VanX, containing Zn(II), Co(II), Fe(II), or Ni(II), catalyzes the hydrolysis of L-alanine-p-nitroanilide producing L-alanine and p-nitroaniline as products; the formation of the latter product (epsilon(404nm) = 10,700 M-1 cm(-1)) can be continuously monitored using UV-VIS spectrophotometry. Zn(II)-, Co(ZI)-, Fe(II)-, and Ni(II)-containing VanX exhibit saturation kinetics when L-alanine-p-nitroanilide is used as the substrate with K-m and k(cat) values ranging from 300 to 700 mu M and 0.028 to 0.080 s(-1), respectively. Inhibition studies using O-[(1S)-aminoethylhydroxyphosphinyl]-D-lactic acid as the inhibitor and L-alanine-p-nitroanilide as the substrate yielded a K-i of 400 +/- 8 mu M at pH 7.0. These studies reveal a continuous assay of VanX activity which could be used to further study the kinetic mechanism of VanX and to allow for the development of high-throughput screening for inhibitors of VanX. (C) 1999 Academic Press.