title: 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.
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-PW(11)O(39))(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 (31)P 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. (15)N-(1)H 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.
authors.affiliation: Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium.
authors.forename: Karen
authors.initials: K
authors.lastname: Stroobants
authors.forename: Eva
authors.initials: E
authors.lastname: Moelants
authors.forename: Hong Giang T
authors.initials: HG
authors.lastname: Ly
authors.forename: Paul
authors.initials: P
authors.lastname: Proost
authors.forename: Kristin
authors.initials: K
authors.lastname: Bartik
authors.forename: Tatjana N
authors.initials: TN
authors.lastname: Parac-Vogt
mesh_terms: D002563:Cerium / Q000737:chemistry*; D002942:Circular Dichroism; D004151:Dipeptides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007768:Lactalbumin / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D009113:Muramidase; D010447:Peptide Hydrolases / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding; D013696:Temperature; D017973:Tungsten Compounds / Q000737:chemistry*; D000099294:Polyoxometalates
chemical_list: D002563:Cerium; D004151:Dipeptides; D007768:Lactalbumin; D009113:Muramidase; D010447:Peptide Hydrolases; D010455:Peptides; D017973:Tungsten Compounds; C102296:polyoxometalate I; D000099294:Polyoxometalates; C421864:hen egg lysozyme