EPR and X-ray crystallographic characterization of the product-bound form of the MnII-loaded methionyl aminopeptidase from Pyrococcus furiosus.
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 A 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.
Copik
Alicja J
AJ
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
Nocek
Boguslaw P
BP
Swierczek
Sabina I
SI
Ruebush
Shane
S
Jang
Se Bok
SB
Meng
Lu
L
D'souza
Ventris M
VM
Peters
John W
JW
Bennett
Brian
B
Holz
Richard C
RC
D000626:Aminopeptidases / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D008345:Manganese / Q000378:metabolism*; D063208:Methionyl Aminopeptidases; D011487:Protein Conformation; D020027:Pyrococcus furiosus / Q000201:enzymology*
D008345:Manganese; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases