A SINGLE ASSAY FOR SIMULTANEOUSLY TESTING EFFECTORS OF ALANINE RACEMASE AND/OR D-ALANINE: D-ALANINE LIGASE

Abstract

The biosynthesis from L-alanine of D-alanyl-D-alanine, required for the peptidoglycan layer of the cell wall of many bacterial species, is catalyzed by two enzymes in series, alanine racemase and D-alanine: D-alanine ligase. A simple in vitro method, called the combined assay, for simultaneously testing for effectors of either or both enzymes in a single assay by coupling these enzymes to each other is described here. The experiments used to derive the optimum conditions for the assay are also described. Each enzyme is included in the assay in rate-limiting amounts, wherein the product of the initial racemase reaction, D-alanine, becomes the substrate for the subsequent ligase. The product of the overall reaction, [¹⁴C]-D-alanyl-D-alanine, is separated chromatographically from the L-[1-¹⁴C]alanine substrate, and from any D-[1-¹⁴C]alanine intermediate, at the end of the incubation, is counted and the percent conversion of substrate to product calculated. The inhibitory effects of 3-fluoro-Dalanine-2d, a known inhibitor of the racemase, and D-cycloserine and DL-1-aminoethylphosphonic acid, inhibitors of both enzymes, were readily detectable. The sensitivity of the combined assay to these inhibitors appears similar to that of earlier assays. This assay has the advantage over previous ones of being able to detect inhibitors of either enzyme in a single assay, thereby avoiding the need to screen each compound in a separate assay of each enzyme.