Molecular Insight from DFT Computations and Kinetic Measurements into the Steric Factors Influencing Peptide Bond Hydrolysis Catalyzed by a Dimeric Zr(IV)-Substituted Keggin Type Polyoxometalate

Peptide bond hydrolysis of several peptides with Gly-X sequence, (X = Gly, Ala, Val, Leu, Ile, Phe) catalyzed by a dimeric Zr(IV)-substituted Keggin type polyoxometalate (POM) (Et2NH2)8[{α-PW11O39Zr(µ-OH)(H2O)}2]·7H2O (1) was studied by means of kinetic experiments and 1H NMR spectroscopy. The observed rate of peptide bond hydrolysis was found to decrease with increase of the side chain bulkiness, from 4.44×10-7 s-1 for Gly-Gly to 0.81×10-7 s-1 for Gly-Ile. A thorough DFT investigation was performed to elucidate (a) the nature of the hydrolytically active species in solution, (b) the mechanism of peptide bond hydrolysis and (c) the influence of the aliphatic residues on the rate of hydrolysis. Formation of substrate-catalyst complexes of the dimeric POM, 1, was predicted as thermodynamically unlikely. Instead, the substrates prefer to bind to the monomerization product of 1, [α-PW11O39Zr(OH)(H2O)]4- (2), which is also present in solution. In the hydrolytically active complex two dipeptide ligands are coordinated to the Zr(IV) center of 2. The first ligand is bidentate bound through its amino nitrogen and amide oxygen atoms while the second one is monodentate bound through a carboxylic oxygen atom. The mechanism of hydrolysis involves nucleophilic attack by a solvent water molecule on the amide carbon atom of the bidentate bound ligand. In this process the uncoordinated carboxylic group of the same ligand acts as a general base to abstract a proton from the attacking water molecule. The decrease of the hydrolysis rate with increase of the side chain bulkiness is mostly due to the increased ligand conformational strain in the rate-limiting transition state, which elevate the reaction activation energy. The conformational strain increases first, upon substitution of Hα in Gly-Gly with aliphatic α-substituent and second, with the β–branching of the α-substituent.status: publishe

Nanozymatic Activity of UiO-66 Metal-Organic Frameworks: Tuning the Nanopore Environment Enhances Hydrolytic Activity toward Peptide Bonds

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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 Trp28-Val29 and Asn44-Arg45 peptide bonds in the presence of a Keggin-type [Ce(α-PW11O39)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 31P 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. 15N-1H 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. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

Chemical Mimics of Aspartate-Directed Proteases: Predictive and Strictly Specific Hydrolysis of a Globular Protein at Asp-X Sequence Promoted by Polyoxometalate Complexes Rationalized by a Combined Experimental and Theoretical Approach

Creating efficient and residue-directed artificial proteases is a challenging task due to the extreme inertness of the peptide bond, combined with the difficulty of achieving specific interactions between the catalysts and the protein side chains. Herein we report strictly site-selective hydrolysis of a multi-subunit globular protein, hemoglobin (Hb) from bovine blood, by a range of ZrIV -substituted polyoxometalates (Zr-POMs) in mildly acidic and physiological pH solutions. Among 570 peptide bonds in Hb, selective cleavage was observed at only eleven sites, each occurring at Asp-X peptide bonds located in the positive patches on the protein surface. The molecular origins of the observed Asp-X selectivity were rationalized by means of molecular docking, DFT-based binding, and mechanistic studies on model peptides. The proposed mechanism of hydrolysis involves coordination of the amide oxygen to ZrIV followed by a direct nucleophilic attack of the side chain carboxylate group on the C-terminal amide carbon atom with formation of a cyclic anhydride, which is further hydrolyzed to give the reaction products. The activation energy for the cleavage of the structurally related Glu-X sequence compared to Asp-X was calculated to be higher by 1.4 kcal mol-1 , which corresponds to a difference of about one order of magnitude in the rates of hydrolysis. The higher activation energy is attributed to the higher strain present in the six-membered ring of glutaric anhydride (Glu-X), as compared to the five-membered ring of the succinic anhydride (Asp-X) intermediate. Similarly, the cleavage at X-Asp and X-Glu bonds are predicted to be kinetically less likely as the corresponding activation energies were 6 kcal mol-1 higher, explaining the experimentally observed selectivity. The synergy between the negatively charged polyoxometalate cluster, which binds at positive patches on protein surfaces, and selective activation of Asp-X peptide bonds located in these regions by ZrIV ions, results in a novel class of artificial proteases with aspartate-directed reactivity, which is very rare among naturally occurring proteases.status: publishe

Molecular Insight from DFT Computations and Kinetic Measurements into the Steric Factors Influencing Peptide Bond Hydrolysis Catalyzed by a Dimeric Zr(IV)-Substituted Keggin Type Polyoxometalate

Peptide bond hydrolysis of several peptides with a Gly-X sequence (X = Gly, Ala, Val, Leu, Ile, Phe) catalyzed by a dimeric Zr­(IV)-substituted Keggin type polyoxometalate (POM), (Et₂NH₂)₈[{α-PW₁₁O₃₉Zr­(μ–OH)­(H₂O)}₂]·7H₂O (<b>1</b>), was studied by means of kinetic experiments and ¹H NMR spectroscopy. The observed rate of peptide bond hydrolysis was found to decrease with increase of the side chain bulkiness, from 4.44 × 10^–7 s^–1 for Gly-Gly to 0.81 × 10^–7 s^–1 for Gly-Ile. A thorough DFT investigation was performed to elucidate (a) the nature of the hydrolytically active species in solution, (b) the mechanism of peptide bond hydrolysis, and (c) the influence of the aliphatic residues on the rate of hydrolysis. Formation of substrate–catalyst complexes of the dimeric POM <b>1</b> was predicted as thermodynamically unlikely. Instead, the substrates prefer to bind to the monomerization product of <b>1</b>, [α-PW₁₁O₃₉Zr­(OH)­(H₂O)]^4– (<b>2</b>), which is also present in solution. In the hydrolytically active complex two dipeptide ligands are coordinated to the Zr­(IV) center of <b>2</b>. The first ligand is bidentate-bound through its amino nitrogen and amide oxygen atoms, while the second ligand is monodentate-bound through a carboxylic oxygen atom. The mechanism of hydrolysis involves nucleophilic attack by a solvent water molecule on the amide carbon atom of the bidentate-bound ligand. In this process the uncoordinated carboxylic group of the same ligand acts as a general base to abstract a proton from the attacking water molecule. The decrease of the hydrolysis rate with an increase in the side chain bulkiness is mostly due to the increased ligand conformational strain in the rate-limiting transition state, which elevates the reaction activation energy. The conformational strain increases first upon substitution of H_α in Gly-Gly with the aliphatic α substituent and second with the β branching of the α substituent

A Zirconium Metal-Organic Framework with SOC Topological Net for Catalytic Peptide Bond Hydrolysis

The discovery of nanozymes for selective cleavage of proteins would boost the emerging areas of modern proteomics, however, the development of efficient and reusable artificial catalysts for peptide bond hydrolysis is challenging. Here we report the detailed catalytic properties of a microporous zirconium carboxylate metal-organic framework, MIP-201, in promoting peptide bond hydrolysis in a simple dipeptide, as well as in horse-heart myoglobin (Mb) protein that consists of 153 amino acids. We demonstrate that MIP-201 features an excellent catalytic activity and selectivity, a good tolerance toward reaction conditions covering a wide range of different pH values, and importantly, an exceptional recycling ability associated with easy regeneration process. Taking into account the excellent catalytic performance of MIP-201 and its other advantages such as 6-connected Zr6 cluster active sites, the green, scalable and cost-effective synthesis, and an outstanding chemical and architectural stability, our finding suggests that MIP-201 may be a promising and practical alternative to the current commercially available catalysts for peptide bond hydrolysis.</div

A zirconium metal-organic framework with SOC topological net for catalytic peptide bond hydrolysis

International audienceThe discovery of nanozymes for selective fragmentation of proteins would boost the emerging areas of modern proteomics, however, the development of efficient and reusable artificial catalysts for peptide bond hydrolysis is challenging. Here we report the catalytic properties of a zirconium metal-organic framework, MIP-201, in promoting peptide bond hydrolysis in a simple dipeptide, as well as in horse-heart myoglobin (Mb) protein that consists of 153 amino acids. We demonstrate that MIP-201 features excellent catalytic activity and selectivity, good tolerance toward reaction conditions covering a wide range of pH values, and importantly, exceptional recycling ability associated with easy regeneration process. Taking into account the catalytic performance of MIP-201 and its other advantages such as 6-connected Zr 6 cluster active sites, the green, scalable and cost-effective synthesis, and good chemical and architectural stability, our findings suggest that MIP-201 may be a promising and practical alternative to commercially available catalysts for peptide bond hydrolysis

Reactivity of Dimeric Tetrazirconium(IV) Wells–Dawson Polyoxometalate toward Dipeptide Hydrolysis Studied by a Combined Experimental and Density Functional Theory Approach

Detailed kinetic studies on the hydrolysis of glycylglycine (Gly-Gly) in the presence of the dimeric tetrazirconium­(IV)-substituted Wells–Dawson-type polyoxometalate Na₁₄­[Zr₄­(P₂W₁₆O₅₉)₂­(μ₃-O)₂­(OH)₂­(H₂O)₄]·57H₂O (<b>1</b>) were performed by a combination of ¹H, ¹³C, and ³¹P NMR spectroscopies. The catalyst was shown to be stable under a broad range of reaction conditions. The effect of pD on the hydrolysis of Gly-Gly showed a bell-shaped profile with the fastest hydrolysis observed at pD 7.4. The observed rate constant for the hydrolysis of Gly-Gly at pD 7.4 and 60 °C was 4.67 × 10^–7 s^–1, representing a significant acceleration as compared to the uncatalyzed reaction. ¹³C NMR data were indicative for coordination of Gly-Gly to <b>1</b> via its amide oxygen and amine nitrogen atoms, resulting in a hydrolytically active complex. Importantly, the effective hydrolysis of a series of Gly-X dipeptides with different X side chain amino acids in the presence of <b>1</b> was achieved, and the observed rate constant was shown to be dependent on the volume, chemical nature, and charge of the X amino acid side chain. To give a mechanistic explanation of the observed catalytic hydrolysis of Gly-Gly, a detailed quantum-chemical study was performed. The theoretical results confirmed the nature of the experimentally suggested binding mode in the hydrolytically active complex formed between Gly-Gly and <b>1</b>. To elucidate the role of <b>1</b> in the hydrolytic process, both the uncatalyzed and the polyoxometalate-catalyzed reactions were examined. In the rate-determining step of the uncatalyzed Gly-Gly hydrolysis, a carboxylic oxygen atom abstracts a proton from a solvent water molecule and the nascent OH nucleophile attacks the peptide carbon atom. Analogous general-base activity of the free carboxylic group was found to take place also in the case of polyoxometalate-catalyzed hydrolysis as the main catalytic effect originates from the −CO···Zr­(IV) binding