Two Continuous Spectrophotometric Assays for Methionine Aminopeptidase

Two spectrophotometric assays have been developed for methionine aminopeptidases (MetAPs). The first method employs a thioester substrate which, upon enzymatic removal of the N-terminal methionine, generates a free thiol group. The released thiol is quantitated using Ellman's reagent. The MetAP reaction is conveniently monitored on a UV-VIS spectrophotometer in a continuous fashion, with the addition of an excess of Ellman's reagent into the assay reaction. Two tripeptide analogues were synthesized and found to be excellent substrates of both Escherichia coli MetAP and human MetAP2 (k(cat)/K(M) = 2.8 x 10(5) M(-1) s(-1) for the most reactive substrate). In the second assay method, the MetAP reaction is coupled to a prolyl aminopeptidase reaction using Met-Pro-p-nitroanilide as substrate. MetAP-catalyzed cleavage of the N-terminal methionine produces prolyl-p-nitroanilide, which is rapidly hydrolyzed by the prolyl aminopeptidase from Bacillus coagulans to release a chromogenic product, p-nitroaniline. This allows the MetAP reaction to be continuously monitored at 405 nm on a UV-VIS spectrophotometer. The assays have been applied to determine the pH optima and kinetic constants for the E. coli and human MetAPs as well as to screen MetAP inhibitors. These results demonstrate that the current assays are convenient, rapid, and sensitive methods for kinetic studies of MetAPs and effective tools for screening MetAP inhibitors.