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Polyoxometalates as a Novel Class of Artificial Proteases: Selective Hydrolysis of Lysozyme under Physiological pH and Temperature Promoted by a Cerium(IV) Keggin-Type Polyoxometalate

Authors

  • Karen Stroobants,

    1. Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Leuven (Belgium), Fax: (+32) 16-3-27992
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  • Eva Moelants,

    1. Department of Microbiology and Immunology, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven (Belgium)
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  • Hong Giang T. Ly,

    1. Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Leuven (Belgium), Fax: (+32) 16-3-27992
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  • Prof. Dr. Paul Proost,

    1. Department of Microbiology and Immunology, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven (Belgium)
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  • Prof. Dr. Kristin Bartik,

    1. Engineering of Molecular Nanosystems, Université libre de Bruxelles, CP 165/64, 50 av. F.D. Roosevelt, 1050 Brussels (Belgium)
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  • Prof. Dr. Tatjana N. Parac-Vogt

    Corresponding author
    1. Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Leuven (Belgium), Fax: (+32) 16-3-27992
    • Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Leuven (Belgium), Fax: (+32) 16-3-27992
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Abstract

Hen-egg-white lysozyme (HEWL) is specifically cleaved at the Trp28–Val29 and Asn44–Arg45 peptide bonds in the presence of a Keggin-type [Ce(α-PW11O39)2]10− polyoxometalate (POM; 1) at pH 7.4 and 37 °C. The reactivity of 1 towards a range of dipeptides was also examined and the calculated reaction rates were comparable to those observed for the hydrolysis of HEWL. Experiments with α-lactalbumin (α-LA), a protein that is structurally highly homologous to HEWL but has a different surface potential, showed no evidence of hydrolysis, which indicates the importance of electrostatic interactions between 1 and the protein surface for the hydrolytic reaction to occur. A combination of spectroscopic techniques was used to reveal the molecular interactions between HEWL and 1 that lead to hydrolysis. NMR spectroscopy titration experiments showed that on protein addition the intensity of the 31P NMR signal of 1 gradually decreased due to the formation of a large protein/polyoxometalate complex and completely disappeared when the HEWL/1 ratio reached 1:2. Circular dichroism (CD) measurements of HEWL indicate that addition of 1 results in a clear decrease in the signal at λ=208 nm, which is attributed to changes in the α-helical content of the protein. 15N–1H heteronuclear single quantum coherence (HSQC) NMR measurements of HEWL in the presence of 1 reveal that the interaction is mainly observed for residues that are located in close proximity to the first site in the α-helical part of the structure (Trp28–Val29). The less pronounced NMR spectroscopic shifts around the second cleavage site (Asn44–Arg45), which is found in the β-strand region of the protein, might be caused by weaker metal-directed binding, compared with strong POM-directed binding at the first site.

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