A New Colorimetric Assay for Methionyl Aminopeptidases: Examination of the Binding of a New Class of Pseudopeptide Analog Inhibitors

Authors

Sanghamitra Mitra, Utah State UniversityFollow
Anna M. Dygas-Holz, Utah State University
Jiri Jiracek, Academy of Sciences of the Czech RepublicFollow
Miroslava Zertova, Academy of Sciences of the Czech Republic
Lenka Zakova, Academy of Sciences of the Czech Republic
Richard C. Holz, Marquette UniversityFollow

Document Type

Article

Publication Date

10-1-2006

Source Publication

Analytical Biochemistry: Methods in the Biological Sciences

Source ISSN

0003-2697

Abstract

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 k_cat and K_m 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 k_cat 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.

Comments

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.

Recommended Citation

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.
https://epublications.marquette.edu/chem_fac/334