A High-Throughput Absorbance-Based Assay for Methionine Produced by Methionine Aminopeptidase Using S-Adenosyl-L-Methionine Synthetase

Abstract

Methionine aminopeptidase (MAP) (E.C. 3.4.11.18) is a metallopeptidase that cleaves the N-terminal methionine (Met) residue from some proteins. MAP is essential for growth of several bacterial pathogens, making it a target for antibacterial drug discovery. MAP enzymes are also present in eukaryotic cells, and one is a target for antiangiogenic cancer therapy. To screen large compound libraries for MAP inhibitors as the starting point for drug discovery, a high-throughput-compatible assay is valuable. Here the authors describe a novel assay, which detects the Met product of MAP-catalyzed peptide cleavage by coupling it to adenosine triphosphate (ATP)–dependent production of S-adenosyl-L-methionine (SAM) and inorganic phosphate (Pi) by SAM synthetase (MetK) combined with inorganic pyrophosphatase. The three Pi ions produced for each Met consumed are detected using Malachite Green/molybdate reagent. This assay can use any unmodified peptide MAP substrate with an N-terminal Met. The assay was used to measure kinetic constants for Escherichia coli MAP using Mn2+ as the activator and the peptide Met-Gly-Met-Met as the substrate, as well as to measure the potency of a MAP inhibitor. A Mn2+ buffer is described that can be used to prevent free Mn2+ depletion by chelating compounds from interfering in screens for MAP inhibitors.