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.

Metal assisted peptide bond hydrolysis: Chemistry, biotechnology and toxicological implications

“…The acceleration factor ranges from 2.3 for Zr(IV)-substituted Lindqvist type polyoxymetalates (Zr(IV)-Ld POMs) to 36 for Ce(IV)-substituted Keggin type polyoxymetalates (Ce(IV)-Kg POMs) [18][19][20]23,24,[27][28][29]31,32]. For Ser-Gly the intermolecular attack of the hydroxyl group on the carbonyl carbon would require the formation of an unfavorable four-membered ring.…”

“…The hydrolysis at the second cleavage site Asn44-Arg45 was mostly directed by the propensity of Ce(IV) to interact with the side chain carboxyl groups. The replacement of Ce(IV) with Zr(IV) or Hf(IV) gave the same pattern of hydrolysis, but lowered the reaction rates [32]. A more detailed study regarding the nature of the interaction of proteins and metal-substituted POMs was conducted using HSA and different types of Zr(IV)-substituted POMs: (i) Wells-Dawsontype POMs, 1:2 Zr(IV)-WD POM and 4:2 Zr(IV)-WD POM, previously described in Section 2.1.4; (ii) Keggin type POMs, the previously described 2:2 Zr(IV)-Kg POM and its analog 1:2 Zr(IV)-Kg POM (Et 2 NH 2 ) 10 The most effective Zr(IV) compounds were Wells-Dawson POMs, 1:2 Zr(IV)-WD POM and 4:2 Zr(IV)-WD POM, with 75 and 50% conversion degrees after 48 h, at pH 7.4 and 60 • C. Less than 35% of HSA was hydrolyzed under the same conditions by other POMs.…”

“…For Gly-Gly, the difference was even more pronounced. Almost a 16-fold reaction rate decrease was observed after the change of hydrolytic compound from 1:2 Zr(IV)-Kg POM to 1:2 Ce(IV)-Kg POM [32].…”

“…Ce(IV)-substituted Keggin-type POM [Ce(␣-PW 11 O 39 ) 2 ] 10− (1:2 Ce(IV)-Kg POM) cleaved dipeptides containing serine residues at C-terminus with rate constants 4.7-10.3 × 10 −7 s −1 at pH 7.0 and 60 • C [32]. It is a reaction much slower than those observed for Ce(IV) salts [23].…”

“…Their hydrolytic activity was examined on various proteins using different POM types described in Section 2.3.1 [32,79,80]. The Zr(IV)-substituted Wells-Dawson type POM (1:2 Zr(IV)-WD POM) seems to be the most promising compound from this group so far, but the possibility of introducing a nearly infinite number of modifications into POMs gives a reasonable hope for obtaining even better artificial proteases.…”