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Molecular Origin of the Hydrolytic Activity and Fixed Regioselectivity of a ZrIV-Substituted Polyoxotungstate as Artificial Protease

Molecular Origin of the Hydrolytic Activity and Fixed Regioselectivity of a ZrIV-Substituted Polyoxotungstate as Artificial Protease

Authors :
Vincent Goovaerts
Gilles Bruylants
Paul Proost
Gregory Absillis
Eva Moelants
Tatjana N. Parac-Vogt
Karen Stroobants
Source :
Chemistry
Publication Year :
2014
Publisher :
Wiley, 2014.

Abstract

A multitechnique approach has been applied in order to identify the thermodynamic and kinetic parameters related to the regioselective hydrolysis of human serum albumin (HSA) promoted by the Wells-Dawson polyoxometalate (POM), K15H[Zr(α2-P2W17O 61)2]. Isothermal titration calorimetry (ITC) studies indicate that up to four POM molecules interact with HSA. While the first interaction site is characterized by a 1:1 binding and an affinity constant of 2×108M-1, the three remaining sites are characterized by a lower global affinity constant of 7×10 5M-1. The higher affinity constant at the first site is in accordance with a high quenching constant of 2.2×108M -1 obtained for fluorescence quenching of the Trp214 residue located in the only positively charged cleft of HSA, in the presence of K 15H[Zr(α2-P2W17O 61)2]. In addition, EuIII luminescence experiments with an EuIII-substituted POM analogue have shown the replacement of water molecules in the first coordination sphere of Eu III due to binding of the metal ion to amino acid side chain residues of HSA. All three interaction studies are in accordance with a stronger POM dominated binding at the positive cleft on the one hand, and interaction mainly governed by metal anchoring at the three remaining positions, on the other hand. Hydrolysis experiments in the presence of K15H[Zr(α 2-P2W17O61)2] have demonstrated regioselective cleavage of HSA at the Arg114-Leu115, Ala257-Asp258, Lys313-Asp314 or Cys392-Glu393 peptide bonds. This is in agreement with the interaction studies as the Arg114-Leu115 peptide bond is located in the positive cleft of HSA and the three remaining peptide bonds are each located near an upstream acidic residue, which can be expected to coordinate to the metal ion. A detailed kinetic study has evidenced the formation of additional fragments upon prolonged reaction times. Edman degradation of the additional reaction products has shown that these fragments result from further hydrolysis at the initially observed cleavage positions, indicating a fixed selectivity for K 15H[Zr(α2-P2W17O 61)2]. Making the cut: A multitechnique approach has been applied to the identification of the thermodynamic and kinetic parameters related to the regioselective hydrolysis of human serum albumin promoted by the Wells-Dawson polyoxometalate (POM), K15H[Zr(α2- P2W17O61)2] (see figure). All interaction studies are in accordance with the observed hydrolysis fragments which are the result of combined POM-dominated binding and interactions governed by metal anchoring. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.<br />SCOPUS: ar.j<br />FLWIN<br />info:eu-repo/semantics/published

Details

ISSN :
09476539
Volume :
20
Database :
OpenAIRE
Journal :
Chemistry - A European Journal
Accession number :
edsair.doi.dedup.....e7e92c078d6422a847d1c549fd09a514
Full Text :
https://doi.org/10.1002/chem.201402683