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

Hen-egg-white lysozyme (HEWL) is specifically cleaved at the Trp28Val29 and Asn44Arg45 peptide bonds in the presence of a Keggin-type [Ce(-PW11O39)2]10 polyoxometalate (POM; 1) at pH7.4 and 37 degrees 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 -lactalbumin (-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 31PNMR 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 =208nm, which is attributed to changes in the -helical content of the protein. 15N1H 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 -helical part of the structure (Trp28Val29). The less pronounced NMR spectroscopic shifts around the second cleavage site (Asn44Arg45), which is found in the -strand region of the protein, might be caused by weaker metal-directed binding, compared with strong POM-directed binding at the first site.