New chromogenic dipeptide substrate for continuous assay of the D-alanyl-D-alanine dipeptidase VanX required for high-level vancomycin resistance.
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.
Anissimova
M
M
Institut de Chimie des Substances Naturelles, CNRS-UPR 2301, 91198 Gif-sur-Yvette, France.
Yaouancq
L
L
Noor
F
F
Badet-Denisot
M-A
MA
Badet
B
B
D001426:Bacterial Proteins / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D002863:Chromogenic Compounds / Q000138:chemical synthesis; D004150:Dipeptidases / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D010636:Phenols / Q000032:analysis; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000032:analysis; D014640:Vancomycin / Q000494:pharmacology; D020713:Vancomycin Resistance
D001426:Bacterial Proteins; D002863:Chromogenic Compounds; D004151:Dipeptides; D004791:Enzyme Inhibitors; D010636:Phenols; D013438:Sulfhydryl Compounds; C482788:alanyl-phenylthioglycine; D014640:Vancomycin; C042983:thiophenol; D004150:Dipeptidases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C091956:VanX dipeptidase