Mechanistic and spectroscopic studies of binuclear metallohydrolases and related model systems

Smith, Sarah (2008). Mechanistic and spectroscopic studies of binuclear metallohydrolases and related model systems PhD Thesis, School of Chemistry & Molecular Bioscience, The University of Queensland.

       
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Author Smith, Sarah
Thesis Title Mechanistic and spectroscopic studies of binuclear metallohydrolases and related model systems
School, Centre or Institute School of Chemistry & Molecular Bioscience
Institution The University of Queensland
Publication date 2008-08
Thesis type PhD Thesis
Supervisor Associate Professor Lawrence Gahan
Dr Gerhard Schenk
Associate Professor Mark Riley
Total pages 298
Total colour pages 40
Total black and white pages 258
Language eng
Subjects 270000 Biological Sciences
Abstract/Summary This thesis examines the role of the metal ions in the binuclear metallohydrolase purple acid phosphatase (PAP), and the properties of model complexes of the PAP active site. The research to date concerning the mechanism and structure of the PAPs is reviewed in Chapter 1. Previous research into structural and functional small molecule mimics of phosphatase enzymes is also discussed. The techniques used throughout this thesis are described in Chapter 2, and include electron paramagnetic resonance (EPR) spectroscopy, magnetic circular dichroism (MCD) spectroscopy, extended X-ray absorption fine structure (EXAFS), mass spectrometry and X-ray crystallography. Chapter 3 describes the structure and spectroscopic and kinetic characterisation of a diiron mimic for the active site of the mammalian PAPs, specifically uteroferrin (Uf). The complex models the key spectroscopic parameters of the enzyme, and shows a similar susceptibility to oxidation in the presence of phosphate; the oxidised phosphate complex is characterised by EPR spectroscopy. The complex is also a functional model of the enzyme. The pH dependence of the hydrolysis of the phosphodiester substrate BDNPP was measured, and resulted in proposal of a reaction mechanism in which a terminally bound Fe(III)-hydroxo is the reaction-initiating nucleophile. A similar study focussing on a binuclear manganese(II,III) complex is described in Chapter 4. This complex is thoroughly characterised by spectroscopic methods, including EPR, variable temperature-variable field (VTVH) MCD and magnetic susceptibility. The complex is weakly antiferromagnetically coupled and the trivalent manganese ion is tetragonally compressed through a Jahn-Teller distortion. The complex is a functional phosphodiesterase, however, the pH dependence is inconsistent with a terminally bound Mn(III)-hydroxo, as observed in the isostructural Fe(III)Fe(II) complex. Instead, a bridging hydroxo nucleophile which is formed upon deprotonation of a terminally Mn(II)-hydroxo species is proposed. The focus of Chapter 5 is a comparison between a Ga(III)Zn(II) model complex and the corresponding derivative of Uf. 71Ga NMR is used to probe the site selectivity ofthe asymmetric ligand. The full site selectivity conferred by the ligand is a good indication that a similar degree of selectivity will be exhibited by the protein. The solution properties of the model complex are consistent with a mechanistic profile similar to that of the diiron complex described in Chapter 3. In contrast, the enzyme shows marked differences to the native diiron system. The metal-metal distance at optimum pH (measured by EXAFS) is shorter than that of the native enzyme, and the pH dependence of the catalytic rate has an unusual shape. Unlike the bell shape observed in the native enzyme, there is an additional shoulder at low pH, and fitting the profile requires a model containing three pKa values. This behaviour is explained in terms of a bridging nucleophile which functions in two different protonation states: hydroxo and oxo. Following on from the unusual pH dependence of the Ga(III)Zn(II) derivative of Uf, in Chapter 6 a selection of metal ion derivatives of Uf are reported, including Fe(III)Zn(II), Al(III)Zn(II), In(III)Zn(II) and Ga(III)Fe(II). Based on the pH dependence of these derivatives, and a detailed review of mechanistically relevant research related to Uf and the PAPs from other sources, a comprehensive mechanism for the PAPs is proposed. This is a ‘one enzyme-two mechanism’ model, in which the identity of the nucleophilic species is dependent upon the source of the enzyme, the metal ion compositions and the substrate. This model is consistent with the evidence presented for all the PAPs to date. The syntheses of three new ligands which improve the degree to which existing ligands mimic the active site of PAP are reported in Chapter 7. All three ligands form heterovalent complexes identified by mass spectrometry. An iron(III) tetramer with a μ4-(OHO)3- core was formed with one of the ligands and crystallographically characterised. This complex dissociates in aqueous conditions to generate a catalytically competent dimeric species. In Chapter 8 a dicopper complex as a model for the non-hydrolytic binuclear metalloenzyme catechol oxidase is reported. The complex is a catalytically competent model and has also been spectroscopically characterised. In contrast to the strongly coupled active site of the enzyme, the copper centres in the model complex are essentially uncoupled, and the metal-metal distance in solution (measured by EPR)indicates that in the solution the complex is flexible. A mechanism differing from that of the enzyme is proposed, whereby the substrate likely binds to only one of the copper centres.
Keyword purple acid phosphatase
uteroferrin
model complex
biomimetic
phosphoesterase
Additional Notes 38, 40, 43, 44, 49, 57, 60, 94, 102-104, 110, 118, 125, 133, 135, 136, 144, 160, 163, 165, 172, 185, 195, 209, 214, 215, 225, 226, 231, 234, 236, 238, 247, 251, 253, 256, 258, 265, 266

 
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Created: Mon, 17 Nov 2008, 18:10:18 EST by Ms Sarah Smith on behalf of Library - Information Access Service