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Article
EPR and X-ray Crystallographic Characterization of the Product-Bound Form of the MnII-Loaded Methionyl Aminopeptidase from Pyrococcus furiosus
Biochemistry
  • Alicja J. Copik, Utah State University
  • Boguslaw P. Nocek, Montana State University - Bozeman
  • Sabina I. Swierczek, Utah State University
  • 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 - Bozeman
  • Brian Bennett, Marquette University
  • Richard C. Holz, Marquette University
Document Type
Article
Language
eng
Format of Original
10 p.
Publication Date
1-1-2005
Publisher
American Chemical Society
Original Item ID
doi: 10.1007/s00775-004-0611-7
Disciplines
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 MnII-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 MnII 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 MnII 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 MnII 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

Biochemistry, Vol. 44, No. 1 (January 2005): 121-129. DOI.

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.

Citation Information
Alicja J. Copik, Boguslaw P. Nocek, Sabina I. Swierczek, Shane Ruebush, et al.. "EPR and X-ray Crystallographic Characterization of the Product-Bound Form of the MnII-Loaded Methionyl Aminopeptidase from Pyrococcus furiosus" Biochemistry (2005) ISSN: 0006-2960
Available at: http://works.bepress.com/richard_holz/88/