Continuous assay for VanX, the D-alanyl-D-alanine dipeptidase required for high-level vancomycin resistance.
Continuous assay for VanX, the D-alanyl-D-alanine dipeptidase required for high-level vancomycin resistance.
复制标题
连续测定 VanX，即高水平万古霉素耐药性所需的 D-丙氨酰-D-丙氨酸二肽酶。
DOI：
10.1006/abio.1999.4166
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Crowder,MW
中科院分区：
文献类型：
--
作者：
Brandt,JJ;Chatwood,LL;Yang,KW;Crowder,MW
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 (ϵ404nm= 10,700 M−1cm−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 Kmand kcatvalues ranging from 300 to 700 μM 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 Kiof 400 ± 8 μM 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.
