Faculty Collaboration Database

Both nucleophile and substrate bind to the catalytic Fe(II)-center in the type-II methionyl aminopeptidase from Pyrococcus furiosus. Inorg Chem 2005 Mar 07;44(5):1160-2

Date

03/01/2005

Pubmed ID

15732944

DOI

10.1021/ic0487934

Scopus ID

2-s2.0-14744298615 (requires institutional sign-in at Scopus site)   20 Citations

Abstract

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.

Author List

Copik AJ, Waterson S, Swierczek SI, Bennett B, Holz RC

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University

MESH terms used to index this publication - Major topics in bold

Aminopeptidases
Binding Sites
Boron Compounds
Catalytic Domain
Electron Spin Resonance Spectroscopy
Iron
Kinetics
Metalloendopeptidases
Nitric Oxide
Pyrococcus furiosus
Spectrum Analysis