EPR and X-ray Crystallographic Characterization of the Product-Bound Form of the Mn^II-Loaded Methionyl Aminopeptidase from Pyrococcus furiosus

Authors

Alicja J. Copik, Utah State University
Boguslaw P. Nocek, Montana State University - BozemanFollow
Sabina I. Swierczek, Utah State UniversityFollow
Shane Ruebush, Utah State University
Se Bok Jang, Montana State University - Bozeman
Lu Meng, Utah State University
Ventris M. D'Souza, Utah State University
John W. Peters, Montana State University - BozemanFollow
Brian Bennett, Marquette UniversityFollow
Richard C. Holz, Marquette UniversityFollow

Document Type

Article

Language

eng

Format of Original

10 p.

Publication Date

1-2005

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Original Item ID

doi: 10.1007/s00775-004-0611-7

Abstract

Methionine aminopeptidases (MetAPs) are ubiquitous metallohydrolases that remove the N-terminal methionine from nascent polypeptide chains. Although various crystal structures of MetAP in the presence of inhibitors have been solved, the structural aspects of the product-bound step has received little attention. Both perpendicular- and parallel-mode electron paramagnetic resonance (EPR) spectra were recorded for the Mn^II-loaded forms of the type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs in the presence of the reaction product l-methionine (l-Met). In general, similar EPR features were observed for both [MnMn(EcMetAP-I)]−l-Met and [MnMn(PfMetAP-II)]−l-Met. The observed perpendicular-mode EPR spectra consisted of a six-line hyperfine pattern at g = 2.03 (A = 8.8 mT) with less intense signals with eleven-line splitting at g = 2.4 and 1.7 (A = 4.4 mT). The former feature results from mononuclear, magnetically isolated Mn^II ions and this signal are 3-fold more intense in the [MnMn(PfMetAP-II)]−l-Met EPR spectrum than in the [MnMn(EcMetAP-I)]−l-Met spectrum. Inspection of the EPR spectra of both [MnMn(EcMetAP-I)]−l-Met and [MnMn(PfMetAP-II)]−l-Met at 40 K in the parallel mode reveals that the [Mn(EcMetAP-I)]−l-Met spectrum exhibits a well-resolved hyperfine split pattern at g = 7.6 with a hyperfine splitting constant of A = 4.4 mT. These data suggest the presence of a magnetically coupled dinuclear Mn^II center. On the other hand, a similar feature was not observed for the [MnMn(PfMetAP-II)]−l-Met complex. Therefore, the EPR data suggest that l-Met binds to [MnMn(EcMetAP-I)] differently than [MnMn(PfMetAP-II)]. To confirm these data, the X-ray crystal structure of [MnMn(PfMetAP-II)]−l-Met was solved to 2.3 Å resolution. Both Mn1 and Mn2 reside in a distorted trigonal bipyramidal geometry, but the bridging water molecule, observed in the [CoCo(PfMetAP-II)] structure, is absent. Therefore, l-Met binding displaces this water molecule, but the carboxylate oxygen atom of l-Met does not bridge between the two Mn^II ions. Instead, a single carboxylate oxygen atom of l-Met interacts with only Mn1, while the N-terminal amine nitrogen atom binds to M2. This l-Met binding mode is different from that observed for l-Met binding [CoCo(EcMetAP-I)]. Therefore, the catalytic mechanisms of type-I MetAPs may differ somewhat from type-II enzymes when a dinuclear metalloactive site is present.

Comments

Accepted version. Biochemistry, Vol. 44, No. 1 (January 2005): 121-129. DOI. © American Chemical Society Publications. Used with permission.

Brian Bennett was affiliated with Medical College of Wisconsin at the time of publication.

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

Recommended Citation

Copik, Alicja J.; Nocek, Boguslaw P.; Swierczek, Sabina I.; Ruebush, Shane; Jang, Se Bok; Meng, Lu; D'Souza, Ventris M.; Peters, John W.; Bennett, Brian; and Holz, Richard C., "EPR and X-ray Crystallographic Characterization of the Product-Bound Form of the Mn^II-Loaded Methionyl Aminopeptidase from Pyrococcus furiosus" (2005). Physics Faculty Research and Publications. 37.
https://epublications.marquette.edu/physics_fac/37