Molecular Discrimination of Type-I over Type-II Methionyl Aminopeptidases

Authors

Krzysztof Swierczek, Utah State University
Alicja J. Copik, Utah State University
Sabina I. Swierczek, Marquette UniversityFollow
Richard C. Holz, Marquette UniversityFollow

Document Type

Article

Publication Date

9-13-2005

Publisher

American Chemical Society Publications

Source Publication

Biochemistry

Source ISSN

0006-2960

Abstract

Two residues that are conserved in type-I methionyl aminopeptidases (MetAPs) but are absent in all type-II MetAPs are the cysteine residues (Escherichia coli MetAP-I:   C59 and C70) that reside at the back of the substrate recognition pocket. These Cys residues are 4.4 Å apart and do not form a disulfide bond. Since bacteria and fungi contain only type-I MetAPs while all human cells contain both type-I and type-II MetAPs, type-I MetAPs represent a novel antibiotic/antifungal target if type-I MetAPs can be specifically targeted over type-II. Based on reaction of the thiol-specific binding reagent 5,5‘-dithio-bis(2-nitrobenzoic acid) (DTNB) with the type-I MetAP from E. coli and the type-II MetAP from Pyrococcus furiosus, the type-I MetAP can be selectively inhibited. Verification that DTNB covalently binds to C59 in EcMetAP-I was obtained by mass spectrometry (MS) from reaction of DTNB with the C59A and C70A mutant EcMetAP-I enzymes. In addition, two inhibitors of EcMetAP-I, 5-iodopentaphosphonic acid (1) and 6-phosphonohexanoic acid (2), were designed and synthesized. The first was designed as a selective-C59 binding reagent while the second was designed as a simple competitive inhibitor of EcMetAP. Indeed, inhibitor 1 forms a covalent interaction with C59 based on activity assays and MS measurements, while 2 does not. These data indicate that type-I MetAPs can be selectively targeted over type-II MetAPs, suggesting that type-I MetAPs represent a new enzymatic target for antibacterial or antifungal agents.

Comments

Accepted version. Biochemistry, Vol. 44, No. 36 (September 13, 2005): 12049-12056. DOI. © 2005 American Chemical Society Publications. Used with permission.

Richard C. Holz was affiliated with Utah State University at the time of publication.

Recommended Citation

Swierczek, Krzysztof; Copik, Alicja J.; Swierczek, Sabina I.; and Holz, Richard C., "Molecular Discrimination of Type-I over Type-II Methionyl Aminopeptidases" (2005). Chemistry Faculty Research and Publications. 316.
https://epublications.marquette.edu/chem_fac/316