title: 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 (P(i)) by SAM synthetase (MetK) combined with inorganic pyrophosphatase. The three P(i) 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 Mn(2+) 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 Mn(2+) buffer is described that can be used to prevent free Mn(2+) depletion by chelating compounds from interfering in screens for MAP inhibitors.
authors.affiliation: AstraZeneca R&D Boston, Waltham, MA 02451, USA. adam.shapiro@astrazeneca.com
authors.forename: Adam B
authors.initials: AB
authors.lastname: Shapiro
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.forename: Jason
authors.initials: J
authors.lastname: Thresher
authors.forename: Grant K
authors.initials: GK
authors.lastname: Walkup
authors.forename: James
authors.initials: J
authors.lastname: Whiteaker
mesh_terms: D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D055808:Drug Discovery / Q000379:methods*; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000592:standards; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008345:Manganese / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism*; D063208:Methionyl Aminopeptidases; D012015:Reference Standards; D012436:S-Adenosylmethionine / Q000096:biosynthesis* / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D008345:Manganese; D012436:S-Adenosylmethionine; D004492:Edetic Acid; D008715:Methionine; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; D008025:Ligases