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A coupled assay measuring Mycobacterium tuberculosis antigen 85C enzymatic activity
Authors:Boucau Julie  Sanki Aditya K  Voss Bradley J  Sucheck Steven J  Ronning Donald R
Institution:a Department of Chemistry, 2801 W. Bancroft Street, University of Toledo, Toledo, OH 43606, USA
b Department of Chemistry, Olivet College, Olivet, MI 49076, USA
Abstract:The prevalence of drug-resistant strains of Mycobacterium tuberculosis (M. tb) emphasizes the need for new antitubercular drugs. An essential component of the drug discovery process is the development of tools to rapidly screen potential drug libraries against important biological targets. Similarly to well-documented M. tb targets, the antigen 85 (Ag85) enzymes are involved in the maintenance of the mycobacterial cell wall. The products synthesized by these mycolyltransferases are the cell wall components most responsible for the reduced permeability of drugs into the bacterial cell, thereby linking Ag85 activity directly with drug resistance. This article presents the development of a high-throughput colorimetric assay suitable for direct monitoring of the enzymatic activity. The assay uses a synthetic substrate containing three chemical moieties: an octanoyl fatty acid, β-d-glucose, and p-nitrophenyl. In the context of the assay, Ag85 catalyzes the removal of the fatty acid and releases p-nitrophenyl-β-d-glucoside. The glucoside is hydrolyzed by β-glucosidase to release the p-nitrophenolate chromophore. With this assay, the KM and kcat values of Ag85C were determined to be 0.047 ± 0.008 mM and 0.062 s−1, respectively. In addition, the assay exhibits a Z′ value of 0.81 ± 0.06, indicating its suitability for high-throughput screening applications and drug development.
Keywords:Acyltransfer  Mycolyltransferase  Antigen 85C  β-Glucosidase  Drug screening  Enzyme-coupled assay
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