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(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 microM 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 microM 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.
Brandt
J J
JJ
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, USA.
Chatwood
L L
LL
Yang
K W
KW
Crowder
M W
MW
D000814:Aniline Compounds; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004150:Dipeptidases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D016984:Enterococcus faecium / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014640:Vancomycin / Q000494:pharmacology
D000814:Aniline Compounds; D001426:Bacterial Proteins; D004151:Dipeptides; D004791:Enzyme Inhibitors; D008670:Metals; D011994:Recombinant Proteins; C004813:alanine-4-nitroanilide; C002956:alanylalanine; D014640:Vancomycin; D004150:Dipeptidases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C091956:VanX dipeptidase