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Molecular Origin of the Hydrolytic Activity and Fixed Regioselectivity of a Zr IV ‐Substituted Polyoxotungstate as Artificial Protease
Author(s) -
Stroobants Karen,
Goovaerts Vincent,
Absillis Gregory,
Bruylants Gilles,
Moelants Eva,
Proost Paul,
ParacVogt Tatja.
Publication year - 2014
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201402683
Subject(s) - isothermal titration calorimetry , chemistry , polyoxometalate , binding constant , quenching (fluorescence) , molecule , titration , hydrolysis , crystallography , stereochemistry , human serum albumin , stability constants of complexes , coordination sphere , binding site , fluorescence , inorganic chemistry , ion , organic chemistry , catalysis , biochemistry , physics , quantum mechanics
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), K 15 H[Zr(α 2 ‐P 2 W 17 O 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 2×10 8 M −1 , the three remaining sites are characterized by a lower global affinity constant of 7×10 5 M −1 . The higher affinity constant at the first site is in accordance with a high quenching constant of 2.2×10 8 M −1 obtained for fluorescence quenching of the Trp214 residue located in the only positively charged cleft of HSA, in the presence of K 15 H[Zr(α 2 ‐P 2 W 17 O 61 ) 2 ]. In addition, Eu III luminescence experiments with an Eu III ‐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 K 15 H[Zr(α 2 ‐P 2 W 17 O 61 ) 2 ] have demonstrated regioselective cleavage of HSA at the Arg114Leu115, Ala257Asp258, Lys313Asp314 or Cys392Glu393 peptide bonds. This is in agreement with the interaction studies as the Arg114Leu115 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 15 H[Zr(α 2 ‐P 2 W 17 O 61 ) 2 ].