1 DOI: 10.1002/chem.201xxxxxx █ Polyoxometalate reactivity Molecular origin of the hydrolytic activity and fixed regioselectivity of a Zr(IV)-substituted polyoxotungstate as artificial protease Karen Stroobants [a] , Vincent Goovaerts [a] , Gregory Absillis [a] , Gilles Bruylants [b] , Eva Moelants [c] , Paul Proost [c] and Tatjana N. Parac-Vogt* [a] Introduction As the unraveling of the structure and function of proteins will lead to a better understanding of biochemical processes occurring in organisms and therefore result in more targeted drug therapy, protein structure determination is of major importance in biomedical research nowadays. Unfortunately, the use of X-ray crystallography, which is the most commonly used method to study the three-dimensional structure of proteins, is limited by the fact that a significant amount of proteins cannot be crystallized. Although this is avoided when NMR spectroscopy is used, in this case a millimolar concentration of a non-aggregated protein solution is needed. Furthermore, only limited success with NMR has been reported for the analysis of fluctuating protein states. Alternative methods study protein fragments rather than the full protein and thus require protein hydrolysis into more manageable peptides which can be traced back to the original protein structure. With a half-life of up to 600 years for peptide bond hydrolysis in the absence of a catalyst, the design of efficient protein hydrolyzing agents is a very challenging task. [1] Furthermore, most biochemical [a] K. Stroobants, V. Goovaerts, Dr. G. Absillis and Prof. Dr. T. N. Parac-Vogt Department of Chemistry KU Leuven Celestijnenlaan 200F, B-3001 Leuven, Belgium Fax: (+)32 16 327992 E-mail: tatjana.vogt@chem.kuleuven.be [b] Dr. G. Bruylants Ecole polytechnique de Bruxelles Université Libre de Bruxelles avenue F.D. Roosevelt 50, B-1050 Bruxelles, Belgium [c] Dr. E. Moelants and Prof. Dr. P. Proost Department of Microbiology and Immunology KU Leuven Minderbroedersstraat 10, B-3000 Leuven, Belgium Supporting information for this article is available on the WWW under http://www.chemeurj.org/ or from the author. Abstract: A multi-technique 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-P2W17O61)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 3·10 6 M -1 , the three remaining sites are characterized by a lower global affinity constant of 6·10 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 K15H[Zr(α2-P2W17O61)2]. In addition, Eu(III) luminescence experiments with an Eu(III) 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(α2-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 K15H[Zr(α2-P2W17O61)2]. This is the peer reviewed version of the following article: Chem. Eur. J. 2014, 20, 9567 – 9577, which has been published in final form at : DOI: 10.1002/chem.201402683. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions."