Both nucleophile and substrate bind to the catalytic Fe(II)-center in the type-II methionyl aminopeptidase from Pyrococcus furiosus.
Metalloproteases utilize their active site divalent metal ions to generate a nucleophilic water/hydroxide. For methionine aminopeptidases (MetAPs), the exact location of this nucleophile, as well as of the substrate, with respect to the active site metal ion is unknown. In order to address this issue, we have examined the catalytically competent Fe(II)-loaded form of PfMetAP-II ([Fe(PfMetAP-II)]) in the absence and presence of both nitric oxide (NO) and the substrate-analogue inhibitor butaneboronic acid (BuBA) by kinetic and spectroscopic (EPR, UV-vis) methods. NO binds to [Fe(PfMetAP-II)] with a Kd of 200 microM forming an {FeNO}7 complex. UV-vis spectra of the resulting [Fe(PfMetAP-II)]-NO complex indicate that the Fe(II) ion is six coordinate. These data suggest that NO binding occurs without displacing the bound aquo/hydroxo moiety in [Fe(PfMetAP-II)]. On the basis of EPR spectra, the resulting Fe-NO complex is best described as NO- (S = 1) antiferromagnetically coupled to a high-spin Fe(III) ion (S = 5/2). The addition of BuBA to [Fe(PfMetAP-II)]-NO displaces the coordinated water molecule forming a six-coordinate adduct. EPR data also indicate that an interaction between the bound NO- and BuBA occurs forming a complex that mimics an intermediate step between the Michaelis complex and the tetrahedral transition-state.
Copik
Alicja J
AJ
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
Waterson
Sarah
S
Swierczek
Sabina I
SI
Bennett
Brian
B
Holz
Richard C
RC
D000626:Aminopeptidases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001896:Boron Compounds; D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D009569:Nitric Oxide; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013057:Spectrum Analysis / Q000379:methods
D001896:Boron Compounds; D009569:Nitric Oxide; C030433:n-butylboronic acid; D007501:Iron; D000626:Aminopeptidases; C106541:methionine aminopeptidase 2; D008666:Metalloendopeptidases