Continuous Assay for VanX, the d-Alanyl–d-Alanine Dipeptidase Required for High-Level Vancomycin Resistance

✍ Scribed by Jeffrey J. Brandt; Lisa L. Chatwood; Ke-Wu Yang; Michael W. Crowder

• Book ID: 102559124
• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 97 KB
• Volume: 272
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4166

✦ Synopsis

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-nitroani- lide producing L-alanine and p-nitroaniline as products; the formation of the latter product (⑀ 404nm ‫؍‬ 10,700 M ؊1 cm ؊1 ) can be continuously monitored using UV-VIS spectrophotometry. Zn(II)-, Co(II)-, 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 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 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.