Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis

An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC. offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (greater than or equal to 10 fold), lower limit of quantitation (LOQ) (0.02 muM versus 1 muM) and wider linear dynamic range (greater than or equal to4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated. (C) 2004 Elsevier B.V. All rights reserved.