Title: Reactivity of oxoanions towards biologically relevant molecules.
Other Titles: Reactiviteit van oxoanionen tegenover biologisch relevante moleculen.
Authors: Ho, Phuong Hien
Issue Date: 22-May-2012
Abstract: Due to their importance in biochemical and chemical applications the chemistry of molybdenum(VI) and vanadium(V) oxo complexes has been intensively investigated. The aim of this study was to explore the reactivity of oxovanadate(V) and oxomolybdate(VI) towards biologically relevant molecules and their model systems such as carboxyesters, peptides, and proteins. Hydrolysis of the carboxyester bond in pNPA was observed in the presence of vanadium(V) oxoanions. In the presence of a mixture of oxovanadates, carboxyester bond hydrolysis in pNPA proceeded under physiological conditions (37 °C, pD 7.4) with a rate constant of 3.0×10-5 s-1 representing an acceleration of at least one order of magnitude in comparison to the uncatalyzed cleavage. The origin of the hydrolytic activity of vanadate is most likely a combination of its nucleophilic nature and the chelating properties which can lead to the stabilization of the transition state. Hydrolysis of the dipeptides glycylserine (Gly-Ser), leucylserine (Leu-Ser), histidylserine (His-Ser), glycylalanine (Gly-Ala), and serylglycine (Ser-Gly) was examined in oxomolybdate solutions by means of 1H, 13C, and 95Mo NMR spectroscopy. In the presence of a mixture of oxomolybdates, the hydrolysis of the peptide bond in Gly-Ser proceeded under neutral pD conditions (pD 7.0, 60 °C) with a rate constant of 5.9×10-6 s-1. The pD dependence of kobs exhibits a bell-shaped profile, with the fastest cleavage observed at pD 7.0. Although the hydrolysis occurred in solutions containing polyoxo-forms of molybdate, the kinetic studies indentified the monomeric molybdate as the hydrolytically active complex. Kinetic experiments at pD 7.0 using a fixed amount of Gly-Ser and increasing amounts of MoO42- resulted in the calculation the catalytic rate constant (k2 = 9.25×10-6 s-1) and the formation constant for the Gly-Ser-MoO42- complex (Kf = 15.25 M-1). The hydrolytic effect of MoO42- can be attributed to its ability to efficiently coordinate X-Ser peptides and polarize their carbonyl group towards the internal nucleophilic attack by the hydroxyl group of the Ser residue. To further explore the biological role of oxovanadates, the reactivity of oxovanadate towards a range of different peptides was examined by means of 1H, 13C, and 51V NMR. In the presence of a mixture of oxovanadates, the hydrolysis of the peptide bond in Gly-Ser proceeded under neutral pD conditions (pD 7.4, 60 °C) with a rate constant of 1.3×10-6 s-1. The pD dependence of kobs exhibits a bell-shaped profile, with the fastest cleavage observed at pD 7.4. The formation constant between vanadate and Gly-Ser, determined by NMR titration experiments in which a fixed amount of Gly-Ser and increasing amounts of vanadate at pD 7.4, was 16.1 M-1. Multinuclear NMR experiments (1H, 13C, and 51V) indicated that monomeric vanadate can form three complexes with Gly-Ser, depending on thepH of solution. While two complexes (Complex 1 and complex 3) have been previously reported, the existence of Complex 2 has not been reported so far. The shifting of 1H and 13C NMR peaks allows a rational design of Complex 2 in which Gly-Ser coordinates to vanadate via its amine nitrogen and amide oxygen, suggesting that this complex is active towards Gly-Ser hydrolysis. The existence of Complex 2 was confirmed by DFT-based molecular modeling. In comparison to the reactivity of MoO42- toward the Gly-Ser hydrolysis, the rate constant in the presence of vanadate was four times slower than that of molybdate. This might be due to the formation of two inactive vanadate-Gly-Ser complexes which lower overall hydrolytic reactivity of vanadate. The hydrolytic activity of oxomolybdate(VI) and oxovanadate(V) towards proteins was examined. Hydrolysis of HEWL with different concentrations of oxovanadate(V) and oxomolybdate(VI) at pH 5.0 was studied by SDS-PAGE and two fragments with a molecular mass of ~12 and 10 kDa were observed. The molecular interaction between oxomolybdate(VI) and oxovanadate(V) and HEWL was studied by CD, 1H-15N HSQC, and 51V NMR spectroscopy. Four binding sites around the amino acid residues Cys6, Thr51, Asp87, and Arg112 which were affected upon the addition of oxomolybdate were determined by means of 1H-15N HSQC. Because Arg112 is solvent accessible and located in a positively charged region of the protein that can stabilize the oxoanion, HEWL cleavage promoted by molybdate and vanadate most probably occurs at this site. The cleavage site at Ser85-Ser86 was also proposed. 51V NMR experiments also indicated that V1, V4, and V10 interact with HEWL. Hydrolysis of other proteins such as OVA, BSA, and HSA promoted by oxomolybdate and oxovanadate was also observed. The appearance of the same cleavage patterns for the hydrolysis of HEWL, OVA, BSA and HSA in the presence of molybdate(VI) and vanadate(V) indicated that these oxoanions exhibit similar selectivity. <w:latentstyles deflockedstate="false" defunhidewhenused="true"  <w:lsdexception="" locked="false" priority="0" semihidden="false"  
ISBN: 978-90-8649-520-7
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Molecular Design and Synthesis
Centre for Surface Chemistry and Catalysis

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