A New Colorimetric Assay for Methionyl Aminopeptidases: Examination of the Binding of a New Class of Pseudopeptide Analog Inhibitors
Analytical Biochemistry: Methods in the Biological Sciences
A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline (l-Met-p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV–Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli (EcMetAP-I) and the type II MetAP from Pyrococcus furiosus (PfMetAP-II). Using l-Met-p-NA, the kinetic constants kcat and Km were determined for EcMetAP-I and PfMetAP-II and were compared with those obtained with a “standard” high-performance liquid chromatography (HPLC) discontinuous assay. The assay has also been used to determine the temperature dependence of the kinetic constant kcat for PfMetAP-II as well as to screen two novel pseudopeptide inhibitors of MetAPs. The results demonstrate that l-Met-p-NA provides a fast, convenient, and effective substrate for both type I and type II MetAPs and that this substrate can be used to quickly screen inhibitors of MetAPs.
Mitra, Sanghamitra; Dygas-Holz, Anna M.; Jiracek, Jiri; Zertova, Miroslava; Zakova, Lenka; and Holz, Richard C., "A New Colorimetric Assay for Methionyl Aminopeptidases: Examination of the Binding of a New Class of Pseudopeptide Analog Inhibitors" (2006). Chemistry Faculty Research and Publications. 334.
ADA accessible version
Accepted version. Analytical Biochemical: Methods in the Biological Sciences, Vol. 357, No. 1 (October 1, 2006): 43-49. DOI. © 2006 Elsevier Inc. Used with permission.
Richard Holz was affiliated with the Utah State University and the Loyola University of Chicago at the time of publication.