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Abstract

Metal-assisted hydrolysis of peptide bond is a promising alternative for enzymatic cleavage of proteins with prospective applications in biochemistry and bioengineering. Many metal ions and complexes have been tested for such reactivity with a number of targets, from dipeptides through oligopeptides through proteins. The majority of reaction mechanisms reported so far is based on the Lewis acidity of a given metal ion. In the alternative hydrolysis reaction the metal ion, Cu(II), Ni(II) or Pd(II), plays a structural role by forming a square planar complex with Ser/Thr-His or Ser/Thr-Xaa-His sequence, which enables a N→O rearrangement of the acyl moiety in the peptide bond downstream from the Ser/Thr residue. Both Lewis acid and N→O acyl rearrangement reaction types are discussed in detail, including molecular mechanisms, the chemical character of hydrolytic agents, reaction conditions, and the origins of differences between the results obtained for peptide and protein models. Toxicological implications and practical applications of metal assisted peptide bond hydrolysis are also presented, with a focus on the Ni(II) assisted N→O acyl rearrangement in Ser/Thr-Xaa-His sequences.

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... In biological systems this reaction is carried out by enzymes with extremely high reaction rates. However, outside biological systems, the hydrolysis of proteins is also an important procedure in areas such as protein structure analysis, protein engineering and protein-cleaving drug design 2,3,4 . Therefore, the high costs and the extreme sensitivity of enzymes to the reaction conditions motivated the development of artificial proteases 2,3 . ...
... However, outside biological systems, the hydrolysis of proteins is also an important procedure in areas such as protein structure analysis, protein engineering and protein-cleaving drug design 2,3,4 . Therefore, the high costs and the extreme sensitivity of enzymes to the reaction conditions motivated the development of artificial proteases 2,3 . The main goal in developing artificial proteases is to achieve adequate reactivity and specificity, as it is very challenging for artificial catalysts to match the exceptional catalytic power of natural enzymes. ...
... Among existing alternatives, homogeneous catalysts based on Lewis acid metal salts often suffer from formation of gels at neutral and basic conditions, leading to loss of reactivity and difficulties in the separation of products and reactants 5 . In comparison, metal complexes prevent the formation of gels during catalysis, but have other shortcomings, such as limited reactivity window, toxicity and poor recyclability 2,5 . More recently metal-substituted polyoxometalate (POM) clusters were developed as homogeneous catalysts for peptide bond hydrolysis. ...
Preprint
div>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
... In biological systems this reaction is carried out by enzymes with extremely high reaction rates. However, outside biological systems, the hydrolysis of proteins is also an important procedure in areas such as protein structure analysis, protein engineering and protein-cleaving drug design 2,3,4 . Therefore, the high costs and the extreme sensitivity of enzymes to the reaction conditions motivated the development of artificial proteases 2,3 . ...
... However, outside biological systems, the hydrolysis of proteins is also an important procedure in areas such as protein structure analysis, protein engineering and protein-cleaving drug design 2,3,4 . Therefore, the high costs and the extreme sensitivity of enzymes to the reaction conditions motivated the development of artificial proteases 2,3 . The main goal in developing artificial proteases is to achieve adequate reactivity and specificity, as it is very challenging for artificial catalysts to match the exceptional catalytic power of natural enzymes. ...
... Among existing alternatives, homogeneous catalysts based on Lewis acid metal salts often suffer from formation of gels at neutral and basic conditions, leading to loss of reactivity and difficulties in the separation of products and reactants 5 . In comparison, metal complexes prevent the formation of gels during catalysis, but have other shortcomings, such as limited reactivity window, toxicity and poor recyclability 2,5 . More recently metal-substituted polyoxometalate (POM) clusters were developed as homogeneous catalysts for peptide bond hydrolysis. ...
Preprint
Full-text available
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.
... Moreover, they often give rise to short fragments, which limits the information retrieved from parent protein structures. Therefore, new chemical approaches for selective protein cleavage are highly desirable, and the development of selective artificial proteases has gained prominence. 9,10 Metal-oxo clusters (MOCs) are a vast class of compounds, with a rich history in inorganic chemistry. 11,12 These clusters have been frequently regarded as discrete soluble intermediates of polymeric metal oxides, and possess extremely attractive physicochemical properties for the development of catalysts, electronic and photochemical devices with a plethora of potential applications in chemistry and technological areas. ...
... These interactions apparently played only a secondary role in these reactions, thus having a quite distinct role from prior artificial metalloproteases based on Cu(II) and Pd(II) complexes that required direct anchoring to the amino acid side chains. 10,35 Moreover, the electrostatic interactions between the M-POM and the amino acid side chains also did not appear to play a relevant role in the hydrolysis of small peptides, although a rate increase was observed in some cases. In view of the promising and rich reactivity with dipeptides, a gradual increase in the complexity of the systems was pursued by examining short oligopeptides 28,36 and the oxidized insulin chain B, an unstructured polypeptide consisting of 30 amino acids. ...
... Moreover, peptide bonds with polar charged side-chain residues were favored over the Lewis basic His and Cys residues that were targeted by other metallic complexes. 10 ...
Article
The selective cleavage of peptide bonds in proteins is of paramount importance in many areas of the biological and medical sciences, playing a key role in protein structure/function/folding analysis, protein engineering, and targeted proteolytic drug design. Current applications that depend on selective protein hydrolysis largely rely on costly proteases such as trypsin, which are sensitive to the pH, ionic strength, and temperature conditions. Moreover, >95% of peptides deposited in databases are generated from trypsin digests, restricting the information within the analyzed proteomes. On the other hand, harsh and toxic chemical reagents such as BrCN are very active but cause permanent modifications of certain amino acid residues. Consequently, transition-metal complexes have emerged as smooth and selective artificial proteases owing to their ability to provide larger fragments and complementary structural information. In the past decade, our group has discovered the unique protease activity of diverse metal-oxo clusters (MOC) and pioneered a distinctive approach to the development of selective artificial proteases. In contrast to classical coordination complexes which often depend on amino acid side chains to control the regioselectivity, the selectivity profile of MOCs is determined by a complex combination of structural factors, such as the protein surface charge, metal coordination to specific side chains, and hydrogen bonding between the protein surface and the MOC scaffold.In this Account, we present a critical overview of our detailed kinetic, spectroscopic, and crystallographic studies in MOC-assisted peptide bond hydrolysis, from its origins to the current rational and detailed mechanistic understanding. To this end, reactivity trends related to the structure and properties of MOCs based on the hydrolysis of small model peptides and key structural aspects governing the selectivity of protein hydrolysis are presented. Finally, our endeavors in seeking the next generation of heterogeneous MOC-based proteases are briefly discussed by embedding MOCs in metal-organic frameworks or using them as discrete nanoclusters in the development of artificial protease-like materials (i.e., nanozymes). The deep and comprehensive understanding sought experimentally and theoretically over the years in aqueous systems with intrinsic polar and charged substrates provides a unique view of the reactivity between inorganic moieties and biomolecules, thereby broadly impacting several different fields (e.g., catalysis in biochemistry, inorganic chemistry, and organic chemistry).
... However, cancer and nickel allergy development mechanisms have not been fully understood. One such mechanism may involve the hydrolysis of a peptide bond preceding the fragment with the Ser/Thr-Xaa-His sequence (where Xaa is any amino acid residue but proline) [7]. Due to hydrolysis, the protein activity is compromised. ...
... Factors that promote cleavage include the presence of the target fragments on flexible and surface-exposed parts of the polypeptide. The cleavage is pH-dependent, and Ni(II) ions break proteins relatively slowly at pH 7.4, but much more rapidly in locations with higher pH [7]. Interestingly, pH values in the mitochondrial matrix are high enough [8][9][10][11][12][13][14][15] to facilitate Ni(II)-assisted hydrolysis of the peptide bond. ...
Article
Full-text available
Nickel is toxic to humans. Its compounds are carcinogenic. Furthermore, nickel allergy is a severe health problem that affects approximately 10–20% of humans. The mechanism by which these conditions develop remains unclear, but it may involve the cleavage of specific proteins by nickel ions. Ni(II) ions cleave the peptide bond preceding the Ser/Thr-Xaa-His sequence. Such sequences are present in all four enzymes of the melatonin biosynthesis pathway, i.e., tryptophan 5-hydroxylase 1, aromatic-l-amino-acid decarboxylase, serotonin N-acetyltransferase, and acetylserotonin O-methyltransferase. Moreover, fragments prone to Ni(II) are exposed on surfaces of these proteins. Our results indicate that all four studied fragments undergo cleavage within tens of hours at pH 8.2 and 37 °C, corresponding with the conditions in the mitochondrial matrix. Since melatonin, a potent antioxidant and anti-inflammatory agent, is synthesized within the mitochondria of virtually all human cells, depleting its supply may be detrimental, e.g., by raising the oxidative stress level. Intriguingly, Ni(II) ions have been shown to mimic hypoxia through the stabilization of HIF-1α protein, but melatonin prevents the action of HIF-1α. Considering all this, the enzymes of the melatonin biosynthesis pathway seem to be a toxicological target for Ni(II) ions.
... In biological systems, this reaction is carried out by enzymes with extremely high reaction rates. Outside of biological systems, the hydrolysis of proteins is an important procedure in areas such as protein-structure analysis, protein engineering, and protein-cleaving drug design [2][3][4] . Therefore, the high costs and the extreme sensitivity of enzymes to the reaction conditions motivated the development of artificial proteases 2,3 , i.e., to achieve adequate reactivity and specificity, as it is very challenging for artificial catalysts to match the exceptional catalytic power of natural enzymes. ...
... Among existing alternatives, homogeneous catalysts based on Lewis acid metal salts often suffer from formation of gels at neutral and basic conditions, leading to loss of reactivity and difficulty in the separating products and reactants 5 . In comparison, metal complexes prevent the formation of gels during catalysis, but have limited reactivity window, toxicity, and poor recyclability 2,5 . More recently, metal-substituted polyoxometalate (POM) clusters were developed as homogeneous catalysts for peptide-bond hydrolysis. ...
Article
Full-text available
The 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.
... The reaction rate depends on pH, temperature and the bulkiness of the first, third and fifth residues, being significantly faster for X 1 = G (fast motifs) . The effectiveness this process was proven for Cu 2+ (Bal et al., 2000) and Pd 2+ ions (Wezynfeld et al., 2016), but Ni-hydrolysis was investigated to the largest extent, due to its higher efficiency. On the other hand, Co 2+ and Zn 2+ ions were proven to be non-reactive in this respect (Bal et al., 2000). ...
... The Ni-hydrolysis of specific susceptible protein motifs is a candidate molecular mechanism of nickel allergy and additional adverse effects of nickel exposure in humans (Wezynfeld et al., 2016). We show that such motifs are present in as many as 40% of human proteins. ...
Article
Full-text available
Deficiency in a principal epidermal barrier protein, filaggrin (FLG), is associated with multiple allergic manifestations, including atopic dermatitis and contact allergy to nickel. Toxicity caused by dermal and respiratory exposures of the general population to nickel-containing objects and particles is a deleterious side effect of modern technologies. Its molecular mechanism may include the peptide bond hydrolysis in X 1 -S/T-c/p-H-c-X 2 motifs by released Ni ²⁺ ions. The goal of the study was to analyse the distribution of such cleavable motifs in the human proteome and examine FLG vulnerability of nickel hydrolysis. We performed a general bioinformatic study followed by biochemical and biological analysis of a single case, the FLG protein. FLG model peptides, the recombinant monomer domain human keratinocytes in vitro and human epidermis ex vivo were used. We also investigated if the products of filaggrin Ni ²⁺ -hydrolysis affect the activation profile of Langerhans cells. We found X 1 -S/T-c/p-H-c-X 2 motifs in 40% of human proteins, with the highest abundance in those involved in the epidermal barrier function, including FLG. We confirmed the hydrolytic vulnerability and pH-dependent Ni ²⁺ -assisted cleavage of FLG-derived peptides and FLG monomer, using in vitro cell culture and ex-vivo epidermal sheets; the hydrolysis contributed to the pronounced reduction in FLG in all of the models studied. We also postulated that Ni-hydrolysis might dysregulate important immune responses. Ni ²⁺ -assisted cleavage of barrier proteins, including FLG, may contribute to clinical disease associated with nickel exposure.
... 1. Different metals can be used as secondary building blocks, as many different metals have been shown catalytic competent in the cleavage of peptide bonds and proteins. [130][131] Initial evidence on the promising reactivity of MOF based on Hf 40 oxo clusters indicates that other tetravalent metals also render MOFs stable enough for the hydrolysis of proteins under physiological-like conditions. Naturally, metals other than tetravalent ones can also be explored. ...
Article
Performing reactions under physiologically relevant conditions often challenges the catalysts’ robustness, reactivity and recyclability. Metal-organic frameworks (MOFs) are an emerging platform for the development of materials with enzyme-like characteristics (i.e., nanozymes), whose applications in bioanalytical devices, biomolecule study, and therapeutics is attracting increasing attention. Despite these promising prospects, developing MOF-based nanozymes that operate in aqueous medium over a broad pH range, and in the presence of a high concentration of salts is frequently challenged by MOFs’ low stability in water, unreliable reactivity, and favorable adsorption of substrates. In this minireview, we share detailed molecular insights on the reactivity of MOFs as nanozymes for hydrolysis of biomolecules, including a number of structure/activity relationships.
... 15,16 Interest in artificial metallopeptidase has been growing. 14,17,18 Based on the understanding of the action of metallopeptidase, complexes of Cu 2+ , 19,20 Pd 2+ , 21 Pt 2+ , 22 and Co 3+ (ref 23) have been investigated for catalytic hydrolysis of amides and peptides under mild conditions. Kita et al. reported Zn acetate and Zn triflate to have higher deamidation activity than Co, Mn, and Cu acetates, while Pd, Ni, and Ag acetates have no activity. ...
Article
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Using DFT calculations and acetamide as the main example, we show that ceria is a potential catalyst for the hydrolysis of amide and similar bonds. The overall reaction is endergonic in the gas phase, yielding acetic acid and ammonia, but is slightly exergonic in the aqueous phase, which facilitates ionization of the products (CH3COO- and NH4 +). Neighboring Ce and O sites on the CeO2(111), (110), and (100) facets are conducive to the formation of an activated metastable tetrahedral intermediate (TI) complex, followed by C-N bond scission. With van der Waals and solvation effects taken into account, the overall reaction energetics is found to be most favorable on the (111) facet as desorption of acetic acid is much more uphill energetically on (110) and (100). We further suggest that the Ce-O-Ce sites on ceria surfaces can activate X(=Y)-Z type bonds in amides, amidines, and carboxylate and phosphate esters, among many others that we term "generalized esters". A Brønsted-Evans-Polanyi relationship is identified correlating the stability of the transition and final states of the X-Z generalized ester bond scission. A simple descriptor (ΣΔχ) based on the electronegativity of the atoms that constitute the bond (X, Y, Z) versus those of the catalytic site (O, Ce, Ce) captures the trend in the stability of the transition state of generalized ester bond scission and suggests a direction for modifying ceria for targeting specific organic substrates.
... Natural hydrolases containing multi-metal centers play important roles in some physiological activities [5], mainly involved in the selectively and efficiently cleavage of the proteins, esters, and DNA [6,7]. Different metal ions or metal complexes containing Zn(II), Co(III), Cu(II), Fe(II/III), Cd(II), Ce(III/IV), Cr(VI), Eu(II), Hf(IV), Mo(IV/VI), Ni(II), Pd(II), Pr(II), Pt(II),V(V), W(IV/VI) and Zr(IV) have been found to be catalytic agents for hydrolysis of unactivated amide bonds [8]. Dinuclear and polynuclear metal complexes are the dominant candidates for metallohydrolases with enzymatic efficiency so far [1][2][3]9]. ...
Article
Full-text available
1H NMR spectroscopy was applied to study the catalytic activity of dinuclear Pd(II)-aqua complexes with different benzodiazine bridging ligands, [{Pd(en)(H2O)}2(μ-qx)]4+ (Pd1), [{Pd(en)(H2O)}2(μ-qz)]4+ (Pd2) and [{Pd(en)(H2O)}2(μ-phtz)]4+ (Pd3) (qx, qz and phtz denote quinoxaline, quinazoline and phthalazine, respectively), in the hydrolytic cleavage of the amide bond in N-acetylated L-methionylglycine (Ac–L–Met–Gly) and L-histidylglycine (Ac–L–His–Gly) dipeptides. All reactions were investigated with an equimolar amount of the reactants at pH = 2.0–2.5 in D2O and at 37 °C. The obtained data for the catalytic activity of Pd1–Pd3 complexes are compared with those previously reported for [{Pt(en)(H2O)}2(μ-L)]4+ (L denotes benzodiazine: qx, qz and phtz), [{Pd(en)(H2O)}2(μ-L)]4+ and [{Pt(en)(H2O)}2(μ-L)]4+ (L denotes diazine: pyrazine and pyridazine) complexes. It was found that catalytic activity of these complexes in peptide cleavage is strongly related to the position of the nitrogen atoms in the benzodiazine or diazine bridging ligand. The investigated dinuclear Pd(II) and Pt(II) complexes show catalytic activity in the selective hydrolysis of the Met–Gly amide bond of Ac–L–Met–Gly dipeptide. Moreover, all the above mentioned Pd(II) complexes were also able to catalyze the regioselective hydrolysis of the His–Gly amide bond of Ac–L–His–Gly dipeptide. However, in the reaction with Ac–L–His–Gly, only Pt(II) aqua complexes containing bridging ligands with two nitrogen atoms in the para-position (quinoxaline and pyrazine) were able to cleave this dipeptide.
... [11][12][13][14] Traditionally, peptide bond hydrolysis in peptides, oliogopeptides and proteins has been achieved by using Lewis acidic metal complexes. [15][16] Our contribution to the field concerns the discovery and development of MOCs unique potential as robust and selective catalysts to cleave the peptide bond. At first, we explored water soluble metal-substituted polyoxometalates (POMs), [17][18][19][20][21] and demonstrated their potential as highly selective proteases. ...
Article
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Proteolytic activity of heterogeneous Zr-based nanozymes is a promising technology for the development of selective protein cleavage protocols which are pivotal in modern proteomics. Here, we report the hydrolytic activity of nanoporous Zr6-based UiO-66 metal-organic framework (MOF) toward peptide bonds in a series of peptides and in hen egg white lysozyme protein. The standard UiO-66 MOF featuring unsubstituted 1,4-benzenedicarboxylate linkers hydrolyzed the glycylglycine with a rate constant of 7.9 × 10-7 s-1, (t1/2 = 10 days), which represents >104-fold acceleration compared to the uncatalyzed reaction. Further, this reactivity was compared with UiO-66 analogs synthesized using modified linkers bearing NO2 and NH2 substituents, or using trifluoracetic acid as a modulator. Although the overall crystalline structure and particle size of these UiO-66 derivatives were generally conserved, they presented distinct nanoporous structures that could be directly correlated with the reaction rates at least an order of magnitude faster than the parent UiO-66 MOF. Further, the modified nanoporous structures also provided distinct reactivities across a series of dipeptide substrates probed. We propose that these differences might arise from the distinct MOF Lewis and Brønsted acidity resulting from the structural modifications. These findings highlight the potential of further optimizing Zr6-based MOF nanozymes to achieve residue-selective hydrolytic activity.
... The latter sequence can be hydrolyzed by Ni(II) ions at the N-terminus of the Ser/ Thr amino acid [2][3][4][5][6][7], and in this way, any kind of C-terminal affinity tags can be easily removed from the expressed protein without leaving any additional amino acids at the termini of the native protein sequence. Such peptide bond cleavage initiated by Ni(II) ions has already found application in the affinity tag removal from fusion proteins [8][9][10]. ...
Article
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Peptide tags are extensively used for affinity purification of proteins. In an optimal case, these tags can be completely removed from the purified protein by a specific protease mediated hydrolysis. However, the interactions of these tags with the target protein may also be utilized for the modulation of the protein function. Here we show that the C-terminal hexahistidine (6 × His) tag can influence the catalytic activity of the nuclease domain of the Colicin E7 metallonuclease (NColE7) used by E. coli to kill competing bacteria under stress conditions. This enzyme non-specifically cleaves the DNA that results in cytotoxicity. We have successfully cloned the genes of NColE7 protein and its R447G mutant into a modified pET-21a DNA vector fusing the affinity tag to the protein upon expression, which would be otherwise not possible in the absence of the gene of the Im7 inhibitory protein. This reflects the inhibitory effect of the 6 × His fusion tag on the nuclease activity, which proved to be a complex process via both coordinative and non-specific steric interactions. The modulatory effect of Zn2+ ion was observed in the catalytic activity experiments. The DNA cleavage ability of the 6 × His tagged enzyme was first enhanced by an increase of metal ion concentration, while high excess of Zn2+ ions caused a lower rate of the DNA cleavage. Modelling of the coordinative effect of the fusion tag by external chelators suggested ternary complex formation instead of removal of the metal ion from the active center.
... Metal ions and complexes that hydrolyze biological molecules have become increasingly important to the fields of chemistry and biology (Grant and Kassai, 2006;Mancin et al., 2012Mancin et al., , 2016Wezynfeld et al., 2016;Yu et al., 2016). The majority of the studies in this area have focused on the reversible addition of water across ribo-and deoxyribonucleic acid phosphodiester bonds and peptide and protein amide bonds. ...
Article
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This report covers major advances in the use of metal ions and complexes to hydrolyze ester and phosphate ester lipid bonds. These metal-based Lewis acids have been investigated as catalysts to isolate fatty acids from biological sources, as probes to study phospholipid bilayer properties, as tools to examine signal transduction pathways, and as lead compounds toward the discovery of therapeutic agents. Metal ions that accelerate phosphate ester hydrolysis under mild conditions of temperature and pH may have the potential to mimic phospholipase activity in biochemical applications.
... This metal complex has potential applications in the field of chemical biology, biochemistry, and bioengineering. A variety of metal ion complexes has been utilized for testing the reactivity with substrates such as peptides, and proteins [67]. Most of the metal catalyzed reactions reported so far are based on the activation of amide carbonyl or water by the Lewis acid mechanism of the metal ion. ...
Article
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Amide bonds are the most prevalent structures found in organic molecules and various biomolecules such as peptides, proteins, DNA, and RNA. The unique feature of amide bonds is their ability to form resonating structures, thus, they are highly stable and adopt particular three-dimensional structures, which, in turn, are responsible for their functions. The main focus of this review article is to report the methodologies for the activation of the unactivated amide bonds present in biomolecules, which includes the enzymatic approach, metal complexes, and non-metal based methods. This article also discusses some of the applications of amide bond activation approaches in the sequencing of proteins and the synthesis of peptide acids, esters, amides, and thioesters.
... Transition metals and their complexes hold great potential as synthetic enzymes. Numerous examples of metal ion promoted peptide bond hydrolysis in peptides, oligopeptides and proteins in aqueous solutions have been reported (Grant and Kassai, 2006;Wezynfeld et al., 2016). However, the selective hydrolysis of proteins in the presence of surfactants has been largely unexplored and only two examples involving Pt II and Pd II complexes have been reported albeit at very acidic pH conditions (2.5-2.9) ...
Article
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In this paper we investigate the effect of three different types of surfactants, on the hydrolysis of Cytochrome c (Cyt c), a predominantly α helical protein containing a heme group, promoted by [Ce(α PW11O39)2]10- (CeK) and [Zr(α PW11O39)2]10- (ZrK) polyoxometalates. In the presence of SDS, Zw3 12, or CHAPS surfactants, which are commonly used for solubilizing hydrophobic proteins, the specificity of CeK or ZrK toward hydrolysis of Cyt c does not change. However, the hydrolysis rate of Cyt c by CeK was increased in the presence of SDS, but decreased in the presence of CHAPS, and was nearly inhibited in the presence of Zw3 12. The Circular dichroism and Tryptophan fluorescence spectroscopy have shown that the structural changes in Cyt c caused by surfactants are similar to those caused by POMs, hence the same specificity in the absence or presence of surfactants was observed. The results also indicate that for Cyt c hydrolysis to occur, large unfolding of the protein is needed in order to accommodate the POMs. While SDS readily unfolds Cyt c, the protein remains largely folded in the presence of CHAPS and Zw3 12. Addition of POMs to Cyt c solutions in CHAPS results in unfolding of the structure allowing the interaction with POMs to occur and results in protein hydrolysis. Zw3 12, however, locks Cyt c in a conformation that resists unfolding upon addition of POM, and therefore results in nearly complete inhibition of protein hydrolysis.
... 80 It plays critical roles as a Lewis acid and in nucleophile activation in the mechanism. 6,13,50,55,62,81,82 As a Lewis acid, it activates the substrate through formation of a bond with the carbonyl oxygen atom. This process enhances the electrophilicity of the carbon atom and polarizes the scissile peptide bond (−C−N−) of the substrate. ...
Article
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IIn this DFT study, activities of 11 different N2O4, N2O3, and NO2 core containing Zr(IV) complexes, 4,13-diaza-18-crown-6 (I'N2O4), 1,4,10-trioxa-7,13-diazacyclopentadecane (I'N2O3) and 2-(2-methoxy)-ethanol (I'NO2), respectively and their analogues in peptide hydrolysis have been investigated. Based on the experimental information, these molecules were created by altering protonation states (singly protonated, doubly protonated or doubly deprotonated) and number of their ligands. The energetics of the I'N2O4, and I'NO2 and their analogues predicted that both stepwise and concerted mechanisms occurred either with similar barriers or the latter was more favorable than the former. They also showed that the doubly deprotonated form hydrolyzed the peptide bond with substantially lower barriers than the barriers for other protonation states. For NO2 core possessing complexes, Zr-(NO2)(OHH)(H2O/OH)n for n = 1-3, the hydroxyl group containing molecules were found to be more reactive than their water ligand possessing counterparts. The barriers for these complexes reduced with an increase in the coordination number (6-8) of the Zr(IV) ion. Among all 11 molecules, the NO2 core possessing and two hydroxyl group containing I'DNO2-2H complex was found to be the most reactive complex with a barrier of 28.9 kcal/mol. Furthermore, barriers of 27.5, 28.9, and 32.0 kcal/mol for hydrolysis of Gly-Glu (negative), Gly-Gly (neutral), and Gly-Lys (positive) substrates, respectively by this complex were in agreement with experiments. The activities of these complexes were explained in terms of basicity of their ligand environment and nucleophilicity of the Zr(IV) center using metal-ligand distances, charge on the metal ion and the metal-nucleophile distance as parameters. These results provide deeper understanding of the functioning of these complexes and will help design Zr(IV)-based synthetic metallopeptidases.
Article
Biointerfaces are significantly affected by electrolytes according to the Hofmeister series. This work reports a systematic investigation on the effect of different metal chlorides, sodium and potassium bromides, iodides and thiocyanates, on the ESI/MS spectra of bovine serum albumin (BSA) in aqueous solution at pH = 2.7. The concentration of each salt was varied to maximize the quality of the ESI/MS spectrum, in terms of peak intensity and bell-shaped profile. The ESI/MS spectra of BSA in the absence and in the presence of salts showed a main protein pattern characterized by the expected mass of 66.5 kDa, except the case of BSA/RbCl (mass 65,3 kDa). In all systems we observed an additional pattern, characterized by at least three peaks with low intensity, whose deconvolution led to suggest the formation of a BSA fragment with a mass of 19.2 kDa. Only NaCl increased the intensity of the peaks of the main BSA pattern, while minimizing that of the fragment. NaCl addition seems to play a crucial role in stabilizing BSA ionized interface against hydrolysis of peptide bonds, through different synergistic mechanisms. To quantify the observed specific electrolyte effects, two “Hofmeister” parameters (Hs and Ps) are proposed. They are obtained using the ratio of (BSA-Salt)/BSA peak intensities for both the BSA main pattern and for its fragment. Synopsis NaCl stabilizes BSA ion and almost prevents fragmentation due to denaturing pH
Article
Controlled protein hydrolysis is an important procedure in proteomics applications, and is used to aid the understanding of protein structure and function. The hydrolysis of hydrophobic proteins is particularly challenging, as due to their poor solubility the use of surfactants, which typically inactivate natural enzymes, is often required. Such limitations of natural enzymes prompted the development of chemical catalysts for the selective hydrolysis of proteins. In this study, the nanozymatic potential of MOF‐808 metal organic framework, has been investigated towards protein hydrolysis in the presence of several surfactants which differ in structure and polarity . The influence of ionic SDS , neutral TX‐100, and zwitterionic Zw3‐12 and CHAPS surfactants has been examined on the hydrolysis of horse heart myoglobin in the presence of MOF‐808. The hydrolysis of myoglobin by MOF‐808 showed that nanozymatic activity of MOF‐808 can be tuned by using appropriate surfactant. To understand the observed reactivity patterns, the interactions between surfactant, MOF and protein were further investigated using a range of spectroscopic methods, which included DLS, NMR, UV‐Vis, CD and Emission Spectroscopy. While the presence of SDS increased the number of observed peptide fragments due to increased protein‐MOF interaction, the use of zwitterionic and neutral surfactant reduced the hydrolytic efficiency, most likely by hindering the efficient protein‐MOF interaction.
Article
The catalytic activity of metal-organic frameworks (MOFs) toward peptides and proteins provides an attractive route for the development of nanozymes for applications in biotechnology and proteomics, particularly in the field of protein identification using mass spectrometry. Here, we report that carefully tuning the Ce/Zr metal ratio is a promising strategy to overcome structural limitations that originate from the high connectivity of the Zr6 node and also increase the peptidase activity of the MOF while preserving the material's nano-topology and stability. A series of bimetallic Ce/Zr-UiO-66 MOFs, in which the amount of Ce was systematically varied from 28 to 87 mol%, have been shown to efficiently catalyze peptide bond hydrolysis in a large variety of peptides with different functional groups, demonstrating their nanozyme potential. Detailed kinetic analysis of the hydrolysis of peptide bonds with a range of Ce/Zr MOFs suggests that among the different metallic clusters present in UiO-66, the Ce6 clusters have superior reactivity compared to the CeZr5 sites. In addition to increasing the catalytic potency of the MOF toward peptide bond hydrolysis, the introduction of Ce(IV) also broadens the reaction scope of MOF catalysts. Selective oxidation of the thiol sidechains and the formation of disulfide bridges have been observed at physiological pH both in cysteine and in glutathione tripeptide as substrates. The rate of oxidation is directly proportional to the amount of Ce present in the MOF, demonstrating that the introduction of Ce into these nanomaterials is a promising strategy to introduce oxidase activity toward biologically relevant substrates. In addition to this, adsorption of dipeptides onto MOF nanomaterials has been studied for the first time. These studies revealed a close link between the nature of peptide side chains and the extent of their adsorption, which has a direct influence on their ability to act as substrates in MOF-catalyzed reactions.
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The 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 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.
Article
Hf-based NU-1000 metal organic framework as a hydrolytic nanozyme toward peptide bonds in dipeptides and hen egg white lysozyme protein revealed its greater stability and better recyclability than previous Zr-/ Hf-based nanozymes. Our results suggest that embedding Hf6O8 oxo-clusters is an efficient strategy to conserve the hydrolytic activity while smoothing the strong substrate adsorption previously observed for a discrete Hf oxo-cluster that hindered further development of their proteolytic potential.
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Copper is one of the most ubiquitous environmental pollutants worldwide. Previous studies have focused on the toxicology of high copper exposure, while there has been comparatively less research on the biological effects of low copper exposure. Low concentrations of copper often exist in freshwater ecosystems, and its impact on the fish is unclear. Both Xenocypris microlepis and Xenocypris davidi are bottom-feeding fishes widely distributed in freshwater ecosystems of China, and they are more likely to be contaminated by low concentrations of copper. Low copper exposure may have effects on molecular regulation at the level of gene expression in the two Xenocypris species. To investigate gene expression differences involved in the response to low copper concentrations between X. microlepis and X. davidi, we established the responses to low copper exposure of 0.01 mg/L for 14 days at the transcriptional level, and RNA-Seq was used to perform a comparative transcriptomic analysis of the liver. A total of 74,135 and 60,894 unigenes from X. microlepis and X. davidi were assembled by transcriptome profiling, respectively. Among these, 84 genes of X. microlepis and 165 genes of X. davidi were identified as differentially expressed genes (DEGs). There were 60 and 135 up-regulated, 24 and 30 down-regulated genes in the two species, respectively. Comparative transcriptome analyses identified five differentially co-expressed genes (DCGs) related to low copper exposure from the DEGs of the two Xenocypris species. The five DCGs were related to the fishes’ growth, antioxidant system, immune system and heavy metal tolerance. The results could help us to understand the molecular mechanisms of the response to low copper exposure, and the data should provide a valuable transcriptomic resource for the genus Xenocypris.
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The capability of ordinary surfactants in solubilizing hydrophobic compounds can come to a limit, if the extension of a contaminant is too large. An attractive goal is the development of surfactants which can actively reduce the size of dirt. Because strong Lewis acids are known to catalyze both bond formation and cleavage, an integration into the surfactant's molecular framework is tempting. End-group functionalized hepta-dentate ligands, which coordinate to metal ions preventing deactivation by hydrolysis over a broad range of pH values while maintaining strong Lewis-acidity, are herein presented. After proof of amphiphilicity and surfactant characteristics, catalytic properties are investigated for different reactions including the cleavage of proteins. The compounds perform better than benchmark catalysts concerning the attack of unreactive amide bonds. A study with two Sc³⁺ species as the active site, one non-amphiphilic, the other one being surface-active, underlines the positive effect of surfactant properties for boosting catalytic efficiency.
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Hydrolysis of amides to carboxylic acids is an industrially important reaction but is challenging due to the difficulty of cleaving the resonance stabilized amidic C–N bond. Twenty-three heterogeneous and homogenous catalysts were examined in the hydrolysis of acetamide. Results showed that Nb2O5 was the most effective heterogeneous catalyst with the greatest yield of acetic acid. A series of Nb2O5 catalysts calcined at various temperatures were characterized and tested in the hydrolysis of acetamide to determine the effects of crystal phase and surface properties of Nb2O5 on catalytic performance. The high catalytic performance observed was attributed mainly to the facile activation of the carbonyl bond by Lewis acid sites that function even in the presence of basic inhibitors (NH3 and H2O). The catalytic studies showed the synthetic advantages of the present method, such as simple operation, catalyst recyclability, additive-free, solvent-free, and wide substrate scope (> 40 examples; up to 95% isolated yield).
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The mechanism responsible for peptide bond hydrolysis by Co(III) and Cu(II) complexes with (oxa)cyclen ligands has been revisited by means of computational tools. We propose that the mechanism starts by substrate coordination and an outer-sphere attack on the amide C atom of a solvent water molecule assisted by the metal hydroxo moiety as a general base, which occurs through six-membered ring transition states. This new mechanism represents a more likely scenario than the previously proposed mechanisms that involved an inner-sphere nucleophilic attack through more strained four-membered rings transition states. The corresponding computed overall free-energy barrier of 25.2 kcal mol-1 for hydrolysis of the peptide bond in Phe-Ala by a cobalt(III) oxacyclen catalyst (1) is consistent with the experimental values obtained from rate constants. Also, we assessed the influence of the nature of the ligand throughout a systematic replacement of N by O atoms in the (oxa)cyclen ligand. Increasing the number of coordinating O atoms accelerates the reaction by increasing the Lewis acidity of the metal ion. On the other hand, the higher reactivity observed for the copper(II) oxacyclen catalyst with respect to the analogous Co(III) complex can be attributed to the larger Brönsted basicity of the copper(II) hydroxo ligand. Ultimately, the detailed understanding of the ligand and metal nature effects allowed us to identify the double role of the metal hydroxo complexes as Lewis acids and Brönsted bases and to rationalize the observed reactivity trends.
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The origin of selectivity in protein hydrolysis promoted by Zr(IV)-substituted polyoxometalates (POMs) has been investigated through a variety of computational techniques. Initially, we analyzed the reaction mechanism for the observed hydrolysis at the Asn44—Arg45 site in the hen egg-white lysozyme protein (HEWL) by the Zr-substituted Lindqvist anion [W5O18Zr(H2O)(OH)]3– (ZrL) using cluster models obtained from molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) calculations, and metadynamics simulations. The mechanism characterization shows that the overall activity is governed by the energy cost to reach the transition state for C–N bond cleavage from the reactants, resulting in a calculated overall free-energy barrier of 121 kJ mol–1 that is in excellent agreement with the values derived from the experimental rate constants (113–134 kJ mol–1). In addition, QM/MM metadynamics simulations on the early stages of the mechanism revealed the formation of an exergonic non-covalent POM···protein complex at the protein surface that was stabilized by positively charged amino acids maintained during the Zr coordination to the amide oxygen. For nonreactive related sites containing Arg (Asn113—Arg114, Arg45—Asn46, and Arg21—Gly22,) we found very similar overall barriers within the cluster model approach (124, 124, and 120 kJ mol–1, respectively); however, their nonbonding POM···protein interactions along the simulated coordination of Zr to the amide oxygen were significantly weaker than those for the reactive Asn44—Arg45 site. Thus, for the HEWL protein the selectivity is governed by an enzyme-like recognition of ZrL at the cleavage site that results in an overall acceleration of the reaction rate compared to those at other sites. Conversely for human serum albumin, (HSA) the observed selectivity was not directed by nonbonding POM···protein interactions but instead was controlled by the protein secondary structure. Calculations on several Arg—Leu sites placed in positive patches showed that peptide bonds in an α-helix structure have higher overall free-energy barriers, while for the active Arg114—Leu115 site in a random coil region the C–N cleavage is facilitated by the extended conformation of the protein chain. All in all, this study has identified and evaluated two complementary factors controlling the selectivity in peptide hydrolysis promoted by transition metal-substituted POMs; hydrolysis is disfavored at α-helical regions of the protein, and then specific positively charged patches can trap the POM via electrostatic-type POM···protein interactions and accelerate the reaction.
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Raman and IR spectroscopic measurements were carried out in formamide (FA) solutions containing variable SnCl4 contents. Pyramidal FA molecules play a major role in these media and are characterised by new bands at 1746, 1190 and 796 cm⁻¹, which may be used as makers. The electrostatic potential (φ) of the coordinated ion shows good relationship with the arising of these bands, which are only seen in the spectra as the cation presents φ ≥ 3.9 × 10²⁰ J C⁻¹. Pyramidalisation of FA is due to the presence of a quaternary N in the [Sn(FA)2Cl2]²⁺ complex, which is formed along with [SnCl6]²⁻. The major Sn(IV)-based complexes are structurally similar to those commonly identified in aqueous medium.
Article
Mono- and dinuclear Cu(II) complexes with Ac-PTVHNEYH-NH2 (L1) and Ac-NHHTLND-NH2 (L2) peptides from FomA protein of Fusobacterium nucleatum were studied by potentiometry, spectroscopic methods (UV-Vis, CD, EPR) and MS technique. The dominant mononuclear complexes for L1 ligand are: CuHL (pH range 5.0-6.0) with 2N {2Nim}, CuH-2L (pH range 8.0-8.5) and CuH-3L species (above pH 9.0) with 4N {Nim, 3N-} coordination modes. The complexes: CuH-1L with 3N {2Nim, N-}, CuH-2L with 3N {Nim, 2N-} and CuH-3L with 4N {Nim, 3N-} binding sites are proposed for the L2 ligand. Probably in the CuH-2L complex for CuL2 system the second His residue in His-His sequence is bound to Cu(II) ion, while the first His residue may stabilize this complex by His-His and/or His-Cu(II) interactions. The dominant dinuclear Cu2L1 complexes in the pH range 6.5-10.5 are: the Cu2H-4L and Cu2H-6L species with 3N{Nim, 2N-}4N{Nim, 3N-} and 4N{Nim, 3N-}4N{Nim, 3N-} binding sites, respectively. In the case of the Cu2L2 complex in the pH range 7.2-10.5, the Cu2H-4L and Cu2H-7L species dominate with 2N{Nim, N-}4N{Nim, 3N-} and (Cu(OH)42-4N{Nim, 3N-}) coordination modes, respectively. The ability to generate reactive oxygen species (ROS) by uncomplexed Cu(II) ions, ligands and their complexes at pH 7.4 in the presence of hydrogen peroxide or ascorbic acid was studied. UV-Vis, luminescence, EPR spin trapping and gel electrophoresis methods were used. Both complexes produce higher level of ROS compared to those of their ligands. ROS produced by Cu(II) complexes are hydroxyl radical and singlet oxygen, which contribute to oxidative DNA cleavage.
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The hydrolysis of hydrophobic or unstructured proteins is challanging due to their poor solubility which often requires presence of surfactants. Here we show that Zr(IV)‐substituted Keggin polyoxometalate (Et 2 NH 2 ) 10 [Zr(α‑PW 11 O 39 ) 2 ] ( 1 ) is able to selectively hydrolyse β‐casein, an intrinsically unstructured protein at pH = 7.4 and 60 o C. Four surfactants which differ in the nature of their polar groups, were investigated for their role in influencing the selectivity and efficiency of protein hydrolysis. Under experimental conditions β‐casein forms micellar structures, in which hydrophilic part of the protein is water accessible and able to interact with 1 . The identical fragmentation pattern of β‐casein in the presence of 1 was observed on SDS‐PAGE both in the presence and absence of surfactants, but the rate of hydrolysis varied depending on the nature of surfactant. While TX‐100 surfactant, which has neutral polar head, caused only slight decrease of hydrolysis rate, stronger inhibition was seen in the presence surfactants having charges in their polar heads . These results were consistent with Trp fluorescence quenching studies which showed that the binding between β‐casein and 1 decreased with the increase in repulsion between the POM and the polar heads of the surfactants. In all the cases, the micellar structure was not significantly affected by the presence of POM or surfactants as indicated by CD spectroscopy.
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NiO nanoparticles and non-stoichiometric black NiO were shown to be effective sources of Ni ²⁺ ions causing sequence-selective peptide bond hydrolysis. NiO nanoparticles were as effective in this reaction as their...
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Mononuclear Pd(II) complexes with two leaving groups are able to promote His-, Cys- and Met-orientated peptide hydrolysis, and exploring the peptide cleavage behavior of novel Pd(II) complex may provide “Omics” studies promising artificial protease. In this study, a novel binuclear Pd(II) complex [Pd2(-O-L-H)(-Cl)](ClO4)2 (L=2,6-bis(N-2'-aminoethylaminomethyl)-p-cresol) was constructed to promote peptide hydrolysis. Although each Pd(II) center has only one leaving group (Cl) in this complex, electrophoresis and LC-MS-MS determination discloses that this complex enables myoglobin cleavage on the second upstream peptide bond from His and Met. Study on peptide celavage confirms also the His- and Met-orientated peptide hydrolysis, yet no Cys-orientated hydrolysis was observed, although the cysteine-induced peptide/complex binding is distinct. Cysteine in peptide even prevents the complex from His-orientated hydrolysis, and oxidizing cysteine residue recovers the His-orientated hydrolysis. This peptide cleavage behavior is quite different from the simultaneous His-, Cys-, and Met-orientated hydrolysis promoted by mononuclear Pd(II) complex. Theoretic study suggests that the two Pd(II) centers of this complex might promote His- and Met-orientated hydrolysis in a synergic manner: one Pd(II) center binds selectively on peptide or protein, and the other coordinates with amide bond and water favoring nucleophilic attack to peptide bond. The thiol group of cysteine is inclined to bridge the two Pd(II) centers to form a “closed” sulphur-bridge structure, disfavoring the Cys-orientated hydrolysis. This study not only demonstrats the peptide cleavage behavior of this binuclear Pd(II) complex, but also provides a polynuclear strategy to regulate the peptide cleavage behavior of Pd(II) complexes.
Chapter
This chapter describes intermolecular catalysis and reactions, intramolecular catalysis and neighbouring group participation, and biologically significant reactions of carboxylic, phosphoric, and sulfonic acids and their derivatives. Kinetic studies of the reaction of diazodiphenylmethane with a series of 5‐substituted orotic acids permitted evaluation of the ‘ortho effect’ using the Charton model and a comparison with similar data from an earlier study of O‐substituted benzoic acids. The results showed that the ortho‐polar effect for both series was dominant, with only a low significance of the orthosteric effect. Theoretical studies of the uncatalysed esterification of acetyl fluoride, chloride, bromide, and iodide by MeOH in the gas phase have revealed the possible cyclic conformations that may occur during experiments at simple ratios of reactants. The calculated free energy of activation for acetyl chloride, 21 kcal mol‐1, was in good agreement with the experimental data.
Chapter
Infrared (IR) and Raman experiments as well as SERS (Surface Enhanced Raman Scattering) and surface investigations were carried out in order to obtain detailed vibrational information on solution and metal/solution interface chemistry. For the former system, especial attention has been given to metal-catalyzed peptide bond cleavage reactions, in which the research of new and more efficient metal complexes is needed. A methodology based on the shifts of the νCO and νCN modes of a simple amide (formamide/FA) has shown to be useful for the prediction of the catalytic activity of metals and a data collection is presented. For the latter, the adsorption dynamics of different species on a copper electrode, in the presence of chloride anions, and the inhibition efficiency of a specific azole (imidazole/Imid) are analyzed and discussed. A proposal for the adsorption mechanism of Imid is suggested on the basis of potential-dependent spectra.
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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 Zr(IV)‐substituted polyoxometalates (Zr‐POMs). Among 570 peptide bonds, selective cleavage was observed at only 11 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. The proposed mechanism of hydrolysis involves coordination of the amide oxygen to Zr(IV) 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 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 Zr(IV) ion, results in artificial proteases with aspartate‐directed reactivity, which is very rare among naturally occurring proteases.
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Site-specific protein cleavage is essential for many protein-production protocols and typically requires proteases. We report the development of a chemical protein-cleavage method that is achieved through the use of a sequence-specific nickel-assisted cleavage (SNAC)-tag. We demonstrate that the SNAC-tag can be inserted before both water-soluble and membrane proteins to achieve fusion protein cleavage under biocompatible conditions with efficiency comparable to that of enzymes, and that the method works even when enzymatic cleavages fail. SNAC-tags allow for versatile sequence-specific cleavage of soluble and membrane proteins with Ni2+ under biocompatible conditions, bypassing enzymatic cleavage and enabling cleavage in situations where commonly used enzymes fail.
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The role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the genes. Here we describe a method for affinity purification of a metallonuclease with its precisely determined native termini. First, the gene encoding the target protein is inserted into a newly designed cloning site, which contains two self-eliminating BsmBI restriction enzyme sites. As a consequence, the engineered DNA code of Ni(II)-sensitive Ser-X-His-X motif is fused to the 3′-end of the inserted gene followed by the gene of an affinity tag for protein purification purpose. The C-terminal segment starting from Ser mentioned above is cleaved off from purified protein by a Ni(II)-induced protease-like action. The success of the purification and cleavage was confirmed by gel electrophoresis and mass spectrometry, while structural integrity of the purified protein was checked by circular dichroism spectroscopy. Our new protein expression DNA construct is an advantageous tool for protein purification, when the complete removal of affinity or other tags, without any remaining amino acid residue is essential. The described procedure can easily be generalized and combined with various affinity tags at the C-terminus for chromatographic applications.
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In this study, mechanisms of hydrolysis of all four chemically diverse cleavage sites of human serum albumin (HSA) by [Zr(OH)(PW11O39)]⁴⁻ (ZrK) have been investigated using the hybrid two‐layer QM/MM (ONIOM) method. These reactions have been proposed to occur through the following two mechanisms: internal attack (IA) and water assisted (WA). In both mechanisms, the cleavage of the peptide bond in the Cys392‐Glu393 site of HSA is predicted to occur in the rate‐limiting step of the mechanism. With the barrier of 27.5 kcal/mol for the hydrolysis of this site, the IA mechanism is found to be energetically more favorable than the WA mechanism (barrier = 31.6 kcal/mol). The energetics for the IA mechanism are in line with the experimentally measured values for the cleavage of a wide range of dipeptides. These calculations also suggest an energetic preference (Cys392‐Glu393, Ala257‐Asp258, Lys313‐Asp314, and Arg114‐Leu115) for the hydrolysis of all four sites of HSA. © 2018 Wiley Periodicals, Inc.
Article
In this work we demonstrate that the previously described reaction of sequence specific Ni(II)-dependent hydrolytic peptide bond cleavage can be performed in complex metalloprotein molecules, such as the Cys2His2 zinc finger proteins. The cleavage within a zinc finger unit possessing a (Ser/Thr)-X-His sequence is not hindered by the presence of the Zn(II) ions. It results in loss of the Zn(II) ion, oxidation of the SH groups and thus, in a collapse of the functional structure. We show that such natural Ni(II)-cleavage sites in zinc finger domains can be edited out without compromising the DNA binding specificity. Inserting a Ni(II)-susceptible sequence between the edited zinc finger and an affinity tag allows for easy removal of the latter sequence by Ni(II) ions after the protein purification. We have shown that this reaction can be executed even when a metal ion binding N-terminal His-tag is present. The cleavage product maintains the native zinc finger structure involving Zn(II) ions. Mass spectra revealed that a Ni(II) ion remains coordinated to the hydrolyzed protein product through the N-terminal (Ser/Thr)-X-His tripeptide segment. The fact that the Ni(II)-dependent protein hydrolysis is influenced by the Ni(II) concentration, pH and temperature of the reaction provides a platform for novel regulated DNA effector design.
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MOF-808, a Zr(IV)-based metal-organic framework, has been proven to be a very effective heterogeneous catalyst for the hydrolysis of the peptide bond in a wide range of peptides and in hen egg white lysozyme protein. The kinetic experiments with a series of Gly-X dipeptides with varying nature of amino acid side chain have shown that MOF-808 exhibits selectivity depending on the size and chemical nature of the X side chain. Dipeptides with smaller or hydrophilic residues were hydrolyzed faster than those with bulky and hydrophobic residues that lack electron rich functionalities which could engage in favourable intermolecular interactions with the btc linkers. Detailed kinetic studies performed by 1H NMR spectroscopy revealed that the rate of glycylglycine (Gly-Gly) hydrolysis at pD 7.4 and 60 °C was 2.69 × 10-4 s-1 (t½ = 0.72 h), which is more than four orders of magnitude faster compared to the uncatalyzed reaction. Importantly, MOF-808 can be recycled several times without compromising the catalytic activity. A detailed quantum-chemical study combined with experimental data allowed to unravel the role of the {Zr6O8} core of MOF-808 in accelerating Gly-Gly hy-drolysis. A mechanism for the hydrolysis of Gly-Gly by MOF-808 is proposed in which Gly-Gly binds to two Zr(IV) centers of the {Zr6O8} core via the oxygen atom of the amide group and the N-terminus. The activity of MOF-808 was also demonstrated towards the hydrolysis of hen egg white lysozyme, a protein consisting of 129 amino acids. Selective frag-mentation of the protein was observed with 55% yield after 25 hours under physiological pH.
Article
Our recent study (Angew. Chem. Int. Ed. 2015, 54, 7391-7394) has shown that Horse heart myoglobin (HHM) was selectively hydrolyzed by a range of Zr(IV)-substituted polyoxometalates (POMs) under mild conditions. In this study the molecular interactions between the Zr-POM catalysts and HHM were investigated by using a range of complementary techniques that include circular dichroism (CD), UV-Vis spectroscopy, tryptophan fluorescence spectroscopy, 1H NMR and 31P NMR spectroscopy. Trp fluorescence quenching study revealed that among all examined Zr-POMs, the most reactive POM, 2:2 Zr(IV)-Keggin, exhibited the strongest interaction with HHM. 31P NMR studies have shown that this POM dissociates in solution resulting in formation of monomeric 1:1Zr(IV)-Keggin structure, which is a likely catalytically active species. In the presence of Zr(IV)-POMs the HHM does not undergo complete denaturation, as evidenced by CD, UV-Vis, tryptophan fluorescence and 1H NMR spectroscopy. CD spectroscopy showed gradual decrease of the -helical content of HHM upon addition of Zr(IV)-POMs, with and the biggest effect was observed in the presence of large Zr(IV)-Wells-Dawson structure, while small Zr(IV)-Lindqvist POM had smallest influence on the decrease of the -helical content of HHM. In all cases the maintaining of the Soret band at 409 nm in the presence of all examined Zr-POMs indicated that no conformational changes in the protein occurred near the heme.
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This study represents the first example of protein hydrolysis at pH = 7.4 and 60 °C by a metal-substituted polyoxometalate (POM) in the presence of a zwitterionic surfactant. Edman degradation results show that in the presence of 0.5% w/v 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) detergent, a Zr(IV)-substituted Wells–Dawson-type POM, K15H[Zr(α2-P2W17O61)2]·25H2O (Zr1-WD2), selectively hydrolyzes human serum albumin exclusively at peptide bonds involving Asp or Glu residues, which contain carboxyl groups in their side chains. The selectivity and extent of protein cleavage are tuned by the CHAPS surfactant by an unfolding mechanism that provides POM access to the hydrolyzed peptide bonds.
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Due to its beneficial corrosion resistance, stainless steel is widely used in, e.g., biomedical applications, as surfaces in food contact, and for products intended to come into skin contact. Low levels of metals can be released from the stainless steel surface into solution, even for these highly corrosion resistant alloys. This needs to be considered in risk assessment and management. This review aims to compile the different metal release mechanisms that are relevant for stainless steel when used in different biological settings. These mechanisms include corrosion-induced metal release, dissolution of the surface oxide, friction-induced metal release, and their combinations. The influence of important physico-chemical surface properties, different organic species and proteins in solution, and of biofilm formation on corrosion-induced metal release is discussed. Chemical and electrochemical dissolution mechanisms of the surface oxides of stainless steel are presented with a focus on protonation, complexation/ligand-induced dissolution, and reductive dissolution by applying a perspective on surface adsorption of com-plexing or reducing ligands and proteins. The influence of alloy composition, microstructure, route of manufacture, and surface finish on the metal release process is furthermore discussed as well as the chemical speciation of released metals. Typical metal release patterns are summarized.
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Peptide thioesters are very useful in protein chemistry, and chemistry- and biochemistry-based protocols are used for the preparation of thioesters. Among such protocols, only a few biochemistry-based approaches have been use for naturally occurring peptide sequences. The development of chemistry-based protocols applicable to natural sequences remains a challenge, and the development of such methods would be a major contribution to protein science. Here, we describe the preparation of peptide thioesters using innovative methodology that features nickel(II)-mediated alcoholysis of a naturally occurring peptide sequence, followed by O−N and N−S acyl transfers. This protocol involves sequential quadruple acyl transfer, termed SQAT. Notably, the SQAT system consists of sequential chemical reactions that allow naturally occurring peptide sequences to be converted to thioesters without requiring an artificial chemical unit.
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Human alpha-1 antitrypsin (AAT) is an abundant serum protein, present at a concentration of 1.0–1.5 g L−1. AAT deficiency is a genetic disease, manifesting itself with emphysema and liver cirrhosis, due to accumulation of a misfolded AAT mutant in hepatocytes. Lung AAT amount is inversely correlated with chronic obstructive pulmonary disease (COPD), a serious and often deadly condition, with increasing frequency in the aging population. Exposure to cigarette smoke and products of fossil fuel combustion aggravates AAT deficiency and COPD according to mechanisms that are not fully understood. Taking into account that these fumes contain particles that can release nickel to human airways and skin, we decided to investigate interactions of AAT with Ni(II) ions within the paradigm of Ni(II)-dependent peptide bond hydrolysis. We studied AAT protein derived from human blood using HPLC, SDS-PAGE, and mass spectrometry. These studies were aided by spectroscopic experiments on model peptides. As a result, we identified three hydrolysis sites in AAT. Two of them are present in the N-terminal part of the molecule next to each other (before Thr-13 and Ser-14 residues) and effectively form one N-terminal cleavage site. The single C-terminal cleavage is located before Ser-285. The N-terminal hydrolysis was more efficient than the C-terminal one, but both abolished the ability of AAT to inhibit trypsin in an additive manner. Nickel ions bound to hydrolysis products demonstrated an ability to generate ROS. These results implicate Ni(II) exposure as a contributing factor in AAT-related pathologies.
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Nickel is harmful for humans, but molecular mechanisms of its toxicity are far from being fully elucidated. One of such mechanisms may be associated with the Ni(II)-dependent peptide bond hydrolysis, which occurs before Ser/Thr in Ser/Thr-Xaa-His sequences. Human annexins A1, A2, and A8, proteins modulating the immune system, contain several such sequences. To test if these proteins are potential molecular targets for nickel toxicity we characterized the binding of Ni(II) ions and hydrolysis of peptides Ac-KALTGHLEE-am (A1-1), Ac-TKYSKHDMN-am (A1-2), and Ac-GVGTRHKAL-am (A1-3), from annexin A1, Ac-KMSTVHEIL-am (A2-1), and Ac-SALSGHLET-am (A2-2), from annexin A2, and Ac-VKSSSHFNP-am (A8-1), from annexin A8, using UV-vis and CD spectroscopies, potentiometry, isothermal titration calorimetry, HPLC and ESI-MS. We found that at physiological conditions (pH 7.4 and 37 °C) peptides A1-2, A1-3, A8-1, and to some extent A2-2, bind Ni(II) ions sufficiently strongly in 4N complexes and are hydrolyzed at sufficiently high rates to justify the notion that these annexins can undergo nickel hydrolysis in vivo. These results are discussed in the context of specific biochemical interactions of respective proteins. Our results also expand the knowledge about Ni(II) binding to histidine peptides by determination of thermodynamic parameters of this process and spectroscopic characterization of 3N complexes. Altogether, our results indicate that human annexins A1, A2, and A8 are potential molecular targets for nickel toxicity and help design appropriate cellular studies.
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Purification of suitable quantity of homogenous protein is very often the bottleneck in protein structural studies. Overexpression of a desired gene and attachment of enzymatically cleavable affinity tags to the protein of interest made a breakthrough in this field. Here we describe the structure of Galleria mellonella silk proteinase inhibitor 2 (GmSPI-2) determined both by X-ray diffraction and NMR spectroscopy methods. GmSPI-2 was purified using a new method consisting in non-enzymatic His-tag removal based on a highly specific peptide bond cleavage reaction assisted by Ni(II) ions. The X-ray crystal structure of GmSPI-2 was refined against diffraction data extending to 0.98 Å resolution measured at 100 K using synchrotron radiation. Anisotropic refinement with the removal of stereochemical restraints for the well-ordered parts of the structure converged with R factor of 10.57% and Rfree of 12.91%. The 3D structure of GmSPI-2 protein in solution was solved on the basis of 503 distance constraints, 10 hydrogen bonds and 26 torsion angle restraints. It exhibits good geometry and side-chain packing parameters. The models of the protein structure obtained by X-ray diffraction and NMR spectroscopy are very similar to each other and reveal the same β2αβ fold characteristic for Kazal-family serine proteinase inhibitors.
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We report for the first time on the selective hydrolysis of a polypeptide system by a metal-substituted polyoxometalate (POM). Oxidized insulin chain B, a 30 amino acid polypeptide, was selectively cleaved by the Zr(IV)-substituted Wells–Dawson POM, K15H[Zr(α2-P2W17O61)2]·25H2O, under physiological pH and temperature conditions in aqueous solution. HPLC-ESI-MS, LC-MS/MS, MALDI-TOF and MALDI-TOF MS/MS data indicate hydrolysis at the Phe1–Val2, Gln4–His5, Leu6–Cys(SO3H)7, and Gly8–Ser9 peptide bonds. The rate of oxidized insulin chain B hydrolysis (0.45 h−1 at pH 7.0 and 60 °C) was calculated by fitting the integration values of its HPLC-UV signal to a first-order exponential decay function. 1H NMR measurements show significant line broadening and shifting of the polypeptide resonances upon addition of the Zr(IV)-POM, indicating that interaction between the Zr(IV)-POM and the polypeptide takes place in solution. Circular dichroism (CD) measurements clearly prove that the flexible unfolded nature of the polypeptide was retained in the presence of the Zr(IV)-POM. The thermal stability of the Zr(IV)-POM in the presence of the polypeptide chain during the hydrolytic reaction was confirmed by 31P NMR spectroscopy. Despite the highly negative charge of the Zr(IV)-POM, the mechanism of interaction appears to be dominated by a strong metal-directed binding between the positively charged Zr(IV) center and negatively charged amino acid side chains.
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Proteolytic enzymes are key signaling molecules in both normal physiological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the application of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers.
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In previous studies we showed that Ni(II) ions can hydrolytically cleave a peptide bond preceding Ser/Thr in peptides of a general sequence RN-(Ser/Thr)-Xaa-His-Zaa-RC, where RN and RC are any peptide sequences. A peptide library screening, assisted by accurate measurements of reaction kinetics for selected peptides, demonstrated the preference for bulky and aromatic residues at variable positions Xaa and Zaa [A. Krężel, E. Kopera, A.M. Protas, A. Wysłouch-Cieszyńska, J. Poznański, W. Bal, J. Am. Chem. Soc., 132 (2010) 3355-3366]. In this work we used a similar strategy to find out whether the next residue downstream to Zaa may influence the reaction rate. Using an Ac-Gly-Ala-Ser-Arg-His-Zaa-Baa-Arg-Leu-NH2 library, with Zaa and Baa positions containing all common amino acids except of Cys, we found a very strong preference for aromatic residues in both variable positions. This finding significantly limits the range of useful Xaa, Zaa and Baa substitutions, thus facilitating the search for optimal sequences for protein engineering applications [E. Kopera, A. Belczyk-Ciesielska, W. Bal, PLoS One 7 (2012) e36350].
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The third edition of the Handbook of Proteolytic Enzymes is a fully revised and updated major reference work in Elsevier's canon. For the first time the Handbook will be available as an online via Elsevier's ScienceDirect platform as well as a three-volume book. The online version will have the enhanced options including online multimedia, cross-referencing capabilities, integrated online delivery and closer integration with the online MEROPS database of peptidases and their inhibitors. This reference work is intended for university libraries, researchers and students, and will be of great interest to the pharmaceutical and biotechnology companies. The new edition will feature articles on approximately 1000 different proteolytic enzymes written by acknowledged experts in the field. Each article will be a full but concise summary, including details of activity and specificity, structural chemistry, preparation and biological aspects. There are also introductory chapters on peptidase classification and mechanisms and a comprehensive index. The one-stop resource for proteolytic enzymes Contains over 830 chapters Covers new research in therapeutics and drug trials Supplies content written by experts in the field.
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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 (1) were performed by a combination of (1)H, (13)C, and (31)P 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. (13)C NMR data were indicative for coordination of Gly-Gly to 1 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 1 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 1. To elucidate the role of 1 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.
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The present review article highlights recent findings in the reactions between different dinuclear Pt(II) complexes with peptides containing cysteine, methionine and histidine residues. The reactions of {trans-[Pt(NH3)2Cl]2(-X)}2+ and {trans-[Pt(NH3)2(H2O)]2(-X)}4+ type complexes with different bridging ligands (X) (X = pyrazine, 4,4'-bipyridyl and 1,2-bis(4-pyridyl)ethane) with the tripeptide glutathione proceeded in two steps. In the first step, one water or chlorido ligand of the dinuclear Pt(II) complex was substituted by the sulfhydryl group of GSH, while in the second step, the remaining water or chlorido ligand from the dinuclear Pt(II)-peptide complex was replaced by the second molecule of glutathione, finally leading to the formation of the {trans-[Pt(NH3)2(GS)]2(-X)}2+ complex. It was shown that the bridging ligand had an important influence on the reactivity of these complexes with glutathione. No hydrolytic cleavage of any amide bond was observed in the reactions between these complexes and glutathione. However, in reactions performed in acidic media (2.0 < pH < 2.5) between dinuclear Pt(II) complexes with the general formulae {[Pt(L)(H2O)]2(-diazine)}4+ (L is different bidentate coordinated diamine ligands and diazine is a pyrazine- or pyridazine-bridging ligand) and N-acetylated peptides containing L-methionine and L-histidine amino acids in the side chains (Ac-L-Met-Gly, Ac-L-His-Gly and Ac-L-Met-Gly-L-His-GlyNH2), regioselective cleavage of these peptides occurred. The mechanism of these hydrolytic reactions was discussed in relation to the structure of the diazine-bridged Pt(II) complex and the investigated peptides. A systematic summary of these results could contribute to the future design of new dinuclear Pt(II) complexes as potential reagents for regioselective cleavage of peptides and proteins.
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In this paper we present a study of the peptide bond formation reaction catalyzed by ribosome. Different mechanistic proposals have been explored by means of Free Energy Perturbation methods within hybrid QM/MM potentials, where the chemical system has been described by the M06-2X functional and the environment by means of the AMBER force field. According to our results, the most favourable mechanism in the ribosome would proceed through an eight-membered ring transition state, involving a proton shuttle mechanism through the hydroxyl group of the sugar and a water molecule. This transition state is similar to that described for the reaction in solution (J. Am. Chem. Soc. 2013, 135, 8708-8719) but the reaction mechanisms are noticeable different. Our simulations reproduce the experimentally determined catalytic effect of ribosome that can be explained by the different behaviour of the two environments. While the solvent reorganizes during the chemical process involving an entropic penalty, the ribosome is preorganized in the formation of the Michaelis complex and does not suffer important changes along the reaction, dampening the charge redistribution of the chemical system.
Chapter
This chapter discusses the applications of the cyanogens bromide reaction. Cyanogen bromide is capable of cleaving thioethers. The action of cyanogen bromide upon proteins is unique in its selective attack on methionine. The reaction of methionine with cyanogen bromide is greatly facilitated by the strong neighboring group effect exerted by the carboxyl group. Cyanogen bromide is synthesized from bromine and potassium cyanide. The selectivity of the reaction of cyanogen bromide with amino acids depends on pH. The selectivity of the cyanogen bromide reaction is demonstrated by exposure to cyanogen bromide of a standard mixture of amino acids, as is used for calibration purposes in automated amino acid analyzer systems. A number of applications to the general structural elucidation of peptides and proteins are: ribonuclease, rabbit γ-globulin, and chymotrypsin. The reaction is applied in many other instances and is useful in a number of ways, such as in the structural elucidation of peptides and proteins, the detection of multiple forms of enzymes and of oxidized residues of methionine, the preparation of physiologically active peptide fragments, the location of newly introduced cross-linkages in enzymes,and the identification and characterization of fragments from enzyme active sites.
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For fast and easy purification, proteins are typically fused with an affinity tag, which often needs to be removed after purification. Here we present a method for the removal of the affinity tag from the target protein in a single step protocol. The protein VIC_001052 of the coral pathogen Vibrio coralliilyticus ATCC BAA-450 contains a metal ion-inducible autocatalytic cleavage (MIIA) domain. Its coding sequence was inserted into an expression vector for the production of recombinant fusion proteins. Following, the target proteins MalE and mCherry were produced as MIIA-Strep fusion proteins in E. coli. The target proteins could be separated from the MIIA-Strep part simply by the addition of calcium or manganese (II) ions within minutes. The cleavage is not affected in the pH range from 5.0 to 9.0 or at low temperatures (6° C). Autocleavage was also observed with immobilized protein on an affinity column. The protein yield was similar to that achieved with a conventional purification protocol. Copyright © 2015. Published by Elsevier B.V.
Article
SDS-PAGE/Edman degradation and HPLC MS/MS showed that zirconium(IV)-substituted Lindqvist-, Keggin-, and Wells–Dawson-type polyoxometalates (POMs) selectively hydrolyze the protein myoglobin at AspX peptide bonds under mildly acidic and neutral conditions. This transformation is the first example of highly sequence selective protein hydrolysis by POMs, a novel class of protein-hydrolyzing agents. The selectivity is directed by Asp residues located on the surface of the protein and is further assisted by electrostatic interactions between the negatively charged POMs and positively charged surface patches in the vicinity of the cleavage site.
Article
SDS-PAGE/Edman degradation and HPLC MS/MS showed that zirconium(IV)-substituted Lindqvist-, Keggin-, and Wells-Dawson-type polyoxometalates (POMs) selectively hydrolyze the protein myoglobin at AspX peptide bonds under mildly acidic and neutral conditions. This transformation is the first example of highly sequence selective protein hydrolysis by POMs, a novel class of protein-hydrolyzing agents. The selectivity is directed by Asp residues located on the surface of the protein and is further assisted by electrostatic interactions between the negatively charged POMs and positively charged surface patches in the vicinity of the cleavage site. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Article
Hydrolysis of various peptides with β-Co(trien)OH(H2O)2+, I, according to Scheme I has been studied for the purpose of developing an analytical method of sequential degradation of peptides. Initial chelation of peptides with I can be accomplished without much difficulty. Hydrolysis of II occurs fairly smoothly with the majority of the tested peptides. However, several peptide complexes containing proline or amino acids with a free carboxyl group, a potential ligand, strongly resisted the hydrolysis, which might present the most serious problem in this method. A solid-state method which runs the hydrolysis on weak cationic exchange resin, as illustrated in Scheme II, has demonstrated a partial success in the stepwise degradation.
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This paper reports on the synthesis and the X-ray characteristics of the binuclear {[Pt(en)Cl]2(μ-pz)}Cl2 complex (en is ethylenediamine, acting as a bidentate ligand; pz is a bridging pyrazine ligand). This complex was converted into the corresponding aqua complex, {[Pt(en)(H2O)]2(μ-pz)}4+, and 1H NMR spectroscopy was applied for a comparison of its catalytic activities in the hydrolysis of N-acetylated l-methionylglycine (Ac-l-Met-Gly) dipeptide with those for the mononuclear [Pt(en)(H2O)2]2+ complex. The peptide and the corresponding platinum(II) complex were reacted in different molar ratios and all reactions were performed at 2.0 < pH < 2.5 in D2O solvent at 37 °C. The course of these hydrolytic reactions is discussed and the difference in the catalytic ability between the mononuclear and binuclear Pt(II) complexes was correlated with the presence of the different hydrolytically active platinum(II)–peptide complexes formed during their reactions with the Ac-l-Met-Gly dipeptide.
Article
Treatment of [Pt(en)Cl-2] complex with pyridazine lead to the formation of new diplatinum(II) coordination compound {[Pt(en)CIl(2)(mu-pydz))Cl-2, which was characterized by NMR spectroscopy and single-crystal X-ray diffraction. X-ray analysis revealed that the needed support for the pyridazine bridge formation, which in other metal complexes has been mostly provided by additional bridging units coordinated to metal centers, might come from supramolecular interactions such as intermolecular hydrogen bonds. This complex was converted into the corresponding aqua complex, {[Pt(en)(H2O)](2)(mu-pydz))(4+), and 1H NMR spectroscopy was applied for comparison of its catalytic activity with that of the analogous pyrazine-bridged {[Pt(en)(H2O)](2)(P-Pz))(4) complex in the hydrolysis of the N-acetylated L-histidylglycine (Ac-L-His-Gly) and L-methionyl-glycyl-L-histidyl-glycineamide (Ac-L-Met-Gly-L-His-GlyNH(2)). All reactions were performed in the pH range 2.0-2.5 and at 37 degrees C. It was found that although dimerization, in general, improves significantly the hydrolytic potency of Pt(II) complexes, the pyridazine Pt(II) dimer is significantly less active than its pyrazine Pt(II) analog, which is probably due to an increased steric effect exerted in the former complex by the ortho-position of the two nitrogen atoms. Consequently, {[Pt(en)(H2O)](2)(mu-pydz))(4+) only binds to the methionine sulfur atom of the Ac-L-Met-Gly-L-His-GlyNFI(2) peptide and promotes cleavage of amide bond that involves the carboxylic group of methionine. In contrast, the analogous pyrazine Pt(II) dimer reacts with both methionine and histidine residues of this tetrapeptide, promoting cleavage of amide bonds involving carboxylic groups of both of these anchoring amino acids. Considering these results it can be assumed that in the polypeptide containing both methionine and histidine residues the regioselective cleavage of the amide bond involving only the carboxylic group of methionine can be achieved successfully by using the presently investigated pyridazine-bridged Pt(II) complex.
Article
Four binuclear {[Pt(L)Cl](2)(mu-pz)}Cl-2-type complexes have been synthesized and characterized by elemental microanalyses and NMR (H-1 and C-13) spectroscopy (L is ethylenediamine, en; (+/-)-1,2-propylenediamine, 1,2-pn; isobutylenediamine, ibn; trans-(+/-)-1,2-diaminocyclohexane, dach and pz is bridging pyrazine ligand). The chlorido complexes were converted into the corresponding aqua species, {[Pt(L) (H2O)](2)(mu-pz)}(4+), and H-1 NMR spectroscopy was applied to study their reactions with the N-acetylated L-methionylglycine, Ac-L-Met-Gly. The {[Pt(L)(H2O)](2)(mu-pz)}(4+) complex and dipeptide were reacted in 1:1 and 1:2 M ratios, respectively, and all reactions were performed in the pH range 2.0-2.5 and at 37 degrees C. In the reactions with equimolar amounts of the reactants all Pt(II) aqua complexes bind to the methionine side chain of Ac-L-Met-Gly dipeptide and promote the cleavage of the amide bond involving the carboxylic group of methionine. It was found that the amount of hydrolyzed dipeptide strongly depends from the steric bulk of bidentate coordinated diamine ligand L in {[Pt(L)(H2O)](2)(mu-pz)}(4+) complex (en > 1,2-pn > ibn > dach). However, in the reaction with an excess of dipeptide the influence of the nature of diamine ligand L on this hydrolytic process could not be observed due to the fact that slow decomposition of {[Pt(L)(H2O)](2)(mu-pz)}(4+) complex was occured.
Article
The hydrolysis of a series of unactivated dipeptides in the presence of a zirconium(IV)-substituted Lindqvist type polyoxometalate, (Me4N)2[W5O18Zr(H2O)3] (designated as ZrW5), was studied by kinetic experiments and NMR spectroscopy. Among the dipeptides examined, those with the X–Ser amino acid sequence were most effectively hydrolyzed. The kinetics of the hydrolysis of histidylserine (His–Ser) was studied in detail; a rate constant of 95.3 (± 0.1) × 10–7 s–1 (pD 7.4 and 60 °C) in the presence of an equimolar amount of ZrW5 was calculated. The binding of His–Ser to ZrW5 was examined by UV/Vis, 1H, 13C, and 183W NMR spectroscopy, and the data indicate that at physiological pD His–Ser chelates the ZrIV through its imidazole nitrogen, amine nitrogen, and amide carbonyl oxygen. In the presence of ZrW5, the pD profile of kobs is bell-shaped, with a maximum reaction rate at pD 7.5. At high pD values an inactive complex is formed as a result of the deprotonation of the amide nitrogen, resulting in inhibition of His–Ser hydrolysis. The effects of pH, temperature, inhibitors, and ionic strength on the hydrolysis rate constant were also investigated, and a full account of the mechanism of this novel reaction is given.
Article
Circular Dichroism (CD) spectroscopy in the visible region (Vis-CD) is a powerful technique to study metal-protein interactions. It can resolve individual d–d electronic transitions as separate bands and is particularly sensitive to the chiral environment of the transition metals. Modern quantum chemical methods enable CD spectra calculations from which, along with direct comparison with the experimental CD data, the conformations and the stereochemistry of the metal-protein complexes can be assigned. However, a clear understanding of the observed spectra and the molecular configuration is largely lacking. In this study, we compare the experimental and computed Vis-CD spectra of Cu2+-loaded model peptides in square-planar complexes. We find that the spectra can readily discriminate the coordination pattern of Cu2+ bound exclusively to main-chain amides from that involving both main-chain amides and a side-chain (i.e. histidine side chain). Based on the results, we develop a set of empirical rules that relates the appearance of particular Vis-CD spectral features to the conformation of the complex. These rules can be used to gain insight into coordination geometries of other Cu2+ or Ni2+-protein complexes.This article is protected by copyright. All rights reserved.
Article
In the present DFT study, mechanisms of peptide hydrolysis by Co(III)- and Cu(II)-containing complexes of 1,4,7,10-tetraazacyclododecane (cyclen), 1-Co and 1-Cu, respectively, and 1-oxa-4,7,10-triazacyclododecane (oxacyclen), 2-Co and 2-Cu, respectively, and their analogues have been investigated. In addition, the effects of the ligand environment, pendant (an organic group containing a recognition site) and metal ion (Co(III), Cu(II), Ni(II), Zn(II), Cd(II), and Pd(II)), on the energetics of this reaction have been elucidated. The reactant of the 1-Co complex exists in the syn–anti conformation, while that of 1-Cu in the syn–syn form. For both these complexes, stepwise and concerted mechanisms were found to occur with similar barriers. The substitution of one of the nitrogen atoms in the cyclen macrocycle to create oxacyclen should occur at position 10 in the Co(III) case and at position 4 in the Cu(II) case. A comparison between the barriers using the common conformation (syn–anti) of 1-Co and 2-Co showed that both complexes hydrolyze the peptide bond with similar barriers, i.e., 39.8 kcal/mol for the former and 40.1 kcal/mol for the latter. This result is in line with the measured data that suggest that the oxacyclen complex exhibits just four times greater activity than the cyclen complex. The removal of the pendant (−C2H5) group in the Co(III)- and Cu(II)-cyclen complexes (1′-Co and 1′-Cu, respectively) reduced the barriers by 9.3 and 3.0 kcal/mol, respectively. For 1′-Co, the barrier of 30.5 kcal/mol is in agreement with the experimental value of 25.9 kcal/mol for the cleavage of myoglobin at pH 9.0 and 50 °C. The reactants of 1′-Cu, 1′-Zn, 1′-Pd, and 1′-Cd adopt the syn–syn conformation, whereas 1′-Ni and 1′-Co exist in the syn–anti geometry. The barriers for 1′-Ni (triplet spin state), 1′-Cu (doublet spin state), 1′-Cd (singlet spin state), 1′-Co (singlet spin state), and 1′-Zn (singlet spin state) are similar, i.e., 27.2, 29.7, 30.5, 30.5, and 31.9 kcal/mol, respectively, and the highest barrier (41.5 kcal/mol) is computed for 1′-Pd (singlet spin state).
Article
The activity of oxomolybdate(VI) towards hen egg white lysozyme (HEWL) was examined under physiological and slightly acidic pH conditions. Purely hydrolytic cleavage of HEWL in the presence of 10 to 100mM of oxomolybdate(VI) after incubation at pH5.0 and 60°C for 2 to 7days was observed in SDS-PAGE experiments. Four cleavage sites, which all occurred at Asp-X sequences and included the Asp18-Asn19, Asp48-Gly49, Asp52-Trp53 and Asp101-Gly102 peptide bonds, were identified with Edman degradation. The molecular interaction between [MoO4](2-) and HEWL was studied by circular dichroism (CD) and (1)H-(15)N heteronuclear single quantum correlation (HSQC) NMR spectroscopy. CD spectroscopy revealed a significant decrease in the α-helical content of HEWL upon addition of oxomolybdate, while (1)H-(15)N HSQC NMR spectroscopy identified the residues which were most affected upon interaction with [MoO4](2-). (95)Mo NMR measurements, performed on oxomolybdate solutions containing HEWL, identified the monomeric [MoO4](2-) form as active species in the hydrolytic reaction. The hydrolysis of the Asp-Gly model peptide in the presence of oxomolybdate(VI) was studied by (1)H NMR, further supporting a hydrolytic mechanism where polarisation of the carbonyl is followed by internal nucleophilic attack on the Asp residue.
Article
Potentiometry and UV-vis and circular dichroism spectroscopies were applied to characterize Cu(II) coordination to the Ac-GASRHWKFL-NH2 peptide. Using HPLC and ESI-MS, we demonstrated that Cu(II) ions cause selective hydrolysis of the Ala-Ser peptide bond in this peptide and characterized the pH and temperature dependence of the reaction. We found that Cu(II)-dependent hydrolysis occurs solely in 4N complexes, in which the equatorial coordination positions of the Cu(II) ion are saturated by peptide donor atoms, namely, the pyridine-like nitrogen of the His imidazole ring and three preceding peptide bond nitrogens. Analysis of the reaction products led to the conclusion that Cu(II)-dependent hydrolysis proceeds according to the mechanism demonstrated previously for Ni(II) ions ( Kopera , E. ; Krężel , A. ; Protas , A. M. ; Belczyk , A. ; Bonna , A. ; Wysłouch-Cieszyńska , A. ; Poznański , J. ; Bal , W. Inorg. Chem. 2010 , 49 , 6636 - 6645 ). However, the pseudo-first-order reaction rate found for Cu(II) is, on average, 100 times lower than that for Ni(II) ions. The greater ability of Cu(II) ions to form 4N complexes at lower pH partially compensates for this difference in rates, resulting in similar hydrolytic activities for the two ions around pH 7.
Article
Background The possible impact of metal release from coronary artery stents has, with their increased use, become a concern.Objectives To study in vitro metal release in biologically relevant milieu from coronary stents made of different alloys.Materials and methodCoronary stents in common use in a department of cardiology at the time of the study were tested. A previously described in vitro technique was used, whereby the stents were kept in the extraction media for a week. Two different extraction media were used to show the necessity of studying the actual biological surrounding of the implant when metal release is investigated. Metal release was determined with atomic absorption spectrometry.ResultsIn this study, we show metal release from stents after immersion in extraction media of artificial sweat and cysteine solution, as illustrative media.Conclusion Metal release from coronary stents is shown. The magnitude of release is influenced by several factors. The extent to which metal release in vitro has potential biological effects, in terms of elicitation of an allergic reaction or induction of sensitization, in vivo needs to be explored. However, as metal release from an implant in a biologically appropriate medium has been established, better risk assessments in relation to delayed hypersensitivity may be undertaken.
Article
In this study, mechanistic insights into the hydrolysis of an extremely stable tertiary peptide bond (Ser-Pro) in the Ser-Pro-Phe sequence by an artificial enzyme, metal (Pd(2+), Co(2+), or Zn(2+))-β-cyclodextrin (CD) complex, have been provided. In particular, the exact reaction mechanism, the location of CD (number of -CH2 groups downstream from the metal center), conformation of CD (primary or secondary rim of CD facing the substrate), the number of CD (one or two), and the optimum metal ion (Pd(2+), Co(2+), or Zn(2+)) have been suggested using a state-of-the-art hybrid quantum mechanics/molecular mechanics (QM/MM: B3LYP/Amber) approach. The QM/MM calculations suggest that the internal delivery mechanism is the most energetically feasible for the peptide hydrolysis. The inclusion of a CD ring at two CH2 groups downstream from the metal center can provide 3 × 10(5) times acceleration in the activity, while the replacement of Pd(2+) with Co(2+) enhances the rate activity another 3.7 × 10(4) times.
Article
In our previous research we demonstrated the sequence specific peptide bond hydrolysis of the R1-(Ser/Thr)-Xaa-His-Zaa-R2 in the presence of Ni(II) ions. The molecular mechanism of this reaction includes an N-O acyl shift of the R1 group from the Ser/Thr amine to the side chain hydroxyl group of this amino acid. The proposed role of the Ni(II) ion is to establish favorable geometry of the reacting groups. In this work we aimed to find out whether the crucial step of this reaction--the formation of the intermediate ester--is reversible. For this purpose we synthesized the test peptide Ac-QAASSHEQA-am, isolated and purified its intermediate ester under acidic conditions, and reacted it, alone, or in the presence of Ni(II) or Cu(II) ions at pH 8.2. We found that in the absence of either metal ion the ester was quickly and quantitatively (irreversibly) rearranged to the original peptide. Such reaction was prevented by either metal ion. Using Cu(II) ions as CD spectroscopic probe we showed that the metal binding structures of the ester and the final amine are practically identical. Molecular calculations of Ni(II) complexes indicated the presence of steric strain in the substrate, distorting the complex structure from planarity, and the absence of steric strain in the reaction products. These results demonstrated the dual catalytic role of the Ni(II) ion in this mechanism. Ni(II) facilitates the acyl shift by setting the peptide geometry, and prevents the reversal of the acyl shift, by stabilizing subsequent reaction products.
Article
Complexes comprising the Lewis acidic ZrIV metal and protein binding polyoxotungstate ligands of Lindqvist-, Keggin- and Wells-Dawson-type were found to region selectively hydrolyze human serum albumin at four distinct positions. Higher reactivities were found for structures with higher polyoxometalate charges and the cleavage positions were found in protein regions of mixed charge. Both findings suggest an electrostatic nature of the observed reactivity. Polyoxometalates for protein hydrolysis: Complexes comprising the Lewis acidic ZrIV metal and protein binding polyoxotungstate ligands of Lindqvist-, Keggin- and Wells-Dawson-type were found to region selectively hydrolyze human serum albumin at four distinct positions (see illustration). Higher reactivities were found for structures with higher polyoxometalate charges and the cleavage positions were found in protein regions of mixed charge.
Article
A common feature of several classes of intrinsically reactive proteins with diverse biological functions is that they undergo self-catalyzed reactions initiated by an N→O or N→S acyl shift of a peptide bond adjacent to a serine, threonine, or cysteine residue. In this study, we examine the N→O acyl shift initiated peptide-bond hydrolysis at the serine residue on a model compound, glycylserine (GlySer), by means of DFT and ab initio methods. In the most favorable rate-determining transition state, the serine COO(-) group acts as a general base to accept a proton from the attacking OH function, which results in oxyoxazolidine ring closure. The calculated activation energy (29.4 kcal mol(-1) ) is in excellent agreement with the experimental value, 29.4 kcal mol(-1) , determined by (1) H NMR measurements. A reaction mechanism for the entire process of GlySer dipeptide hydrolysis is also proposed. In the case of proteins, we found that when no other groups that may act as a general base are available, the N→O acyl shift mechanism might instead involve a water-assisted proton transfer from the attacking serine OH group to the amide oxygen. However, the calculated energy barrier for this process is relatively high (33.6 kcal mol(-1) ), thus indicating that in absence of catalytic factors the peptide bond adjacent to serine is no longer a weak point in the protein backbone. An analogous rearrangement involving the amide N-protonated form, rather than the principle zwitterion form of GlySer, was also considered as a model for the previously proposed mechanism of sea-urchin sperm protein, enterokinase, and agrin (SEA) domain autoproteolysis. The calculated activation energy (14.3 kcal mol(-1) ) is significantly lower than the experimental value reported for SEA (≈21 kcal mol(-1) ), but is still in better agreement as compared to earlier theoretical attempts.
Article
Today, proteins are typically overexpressed using solubility-enhancing fusion tags that allow for affinity chromatographic purification and subsequent removal by site-specific protease cleavage. In this review, we present an alternative approach to protein production using fusion partners specifically designed to accumulate in insoluble inclusion bodies. The strategy is appropriate for the mass production of short peptides, intrinsically disordered proteins, and proteins that can be efficiently refolded in vitro. There are many fusion protein systems now available for insoluble expression: TrpLE, ketosteroid isomerase, PurF, and PagP, for example. The ideal fusion partner is effective at directing a wide variety of target proteins into inclusion bodies, accumulates in large quantities in a highly pure form, and is readily solubilized and purified in commonly used denaturants. Fusion partner removal under denaturing conditions is biochemically challenging, requiring harsh conditions (e.g., cyanogen bromide in 70% formic acid) that can result in unwanted protein modifications. Recent advances in metal ion-catalyzed peptide bond cleavage allow for more mild conditions, and some methods involving nickel or palladium will likely soon appear in more biological applications.
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The formation of the complexes formed by NiII and ZnII with Asp-Asp-Asp and a series of tetrapeptides containing one or two Asp residues or one Glu residue are reported. Stability constants were measured pH-metrically. The particular species and the metal ion binding sites were determined using 1H NMR, UV-vis and CD spectroscopy. The β-carboxylate group of the Asp residue stabilizes the complexes significantly, particularly when present as the N-terminal residue. As a result the tendency for NiII to deprotonate and bind to amide-nitrogen atoms, forming planar diamagnetic complexes, is reduced and their formation delayed to a significantly higher pH when compared to other peptides. The side chain of the Glu residue has a much smaller effect. As anticipated, ZnII was unable to deprotonate and bind to peptide nitrogens.
Article
In this paper the reactivity of K15H[Zr(α2-P2W17O61)2]·25H2O (), a Zr(iv)-substituted Wells-Dawson polyoxometalate, is examined towards a series of Gly-Aa, Aa-Gly or Aa-Ser dipeptides, in which the nature and the size of the Aa amino acid side chain were varied. The rate of peptide bond hydrolysis, determined by (1)H NMR experiments, in Gly-Aa dipeptides is strongly dependent on the molecular volume and the chemical structure of the Aa side chain. When the volume of the aliphatic side chain of the Aa residue in Gly-Aa increased, a clear decrease in the hydrolysis rate was observed. Replacing one α-H in the C-terminal Gly residue of Gly-Gly by a methyl group (Gly-Ala) resulted in a 6-fold reactivity decrease, pointing towards the importance of steric factors for efficient peptide bond hydrolysis. The rate constants for peptide bond hydrolysis in Gly-Aa dipeptides at pD 5.0 and 60 °C ranged from 208.0 ± 15.6 × 10(-6) min(-1) for Gly-Ser to 5.0 ± 1.0 × 10(-6) min(-1) for Gly-Glu, reflecting the influence of the different nature of the amino acid side chains on the hydrolysis rate. Faster hydrolysis was observed for peptides containing Ser and Thr since the hydroxyl group in their side chain is able to facilitate amide bond hydrolysis by promoting an N→O acyl rearrangement. Peptides containing positively charged side chains at pD 5.0 show enhanced hydrolysis rates as a result of the secondary electrostatic interactions with the negatively charged surface of the polyoxometalate, which stabilize the peptide-polyoxometalate complex. A slow hydrolysis rate was observed for Gly-Glu, because of the preferential coordination of the carboxylate group in the side chain of Glu to Zr(iv), which prevents coordination of the peptide carbonyl group and its activation towards hydrolysis.
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Di- and tri-peptides are efficiently hydrolysed by the cerium(IV)–γ-cyclodextrin complex in neutral and homogeneous solutions.
Article
Chemical cleavage with reactive oxygen species generated by EPD-Fe, a protein-tethered EDTA-Fe reagent, has been proposed as a method to map the structure of nonnative equilibrium protein folding intermediates [Ermacora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., & Fox, R. 0. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387]. The chemical structure of protein cleavage products and the mechanism of backbone scission for this class of reagents have been unclear. Here, we report the nature of EPD-Fe-mediated backbone cleavage of a small model peptide. The EPD-Fe reagent was attached to a partially alpha-helical peptide, alpha1 BAla (Ac-AEAEEAAKKAKEACKA-NH2), through a mixed disulfide. Backbone cleavage was initiated by addition of the iron reductant ascorbate. Chemical analysis of the novel cleavage products revealed an oxidative cleavage mechanism, probably initiated by diffusible hydroxyl radicals. The EPD-Fe-mediated cleavage technique appears to be suitable for the analysis of nonnative protein states such as the molten globule.
Article
The bonding interaction and hydrolytic cleavage of oxidized insulin B chain with ZnCl2 in molar ratios of 1:1 1:2 and 1:3 at pH value of 2.5 and 40 °C were investigated by electrospray ionization mass spectrometry and tandem mass spectrometry. The results show that the binding sites of Zn 2+ with oxidized insulin B chain are Arg22 and imidazole groups of His5, His10, which leads to the selective cleavages of the peptide bonds at Asn3-Gln4, His5-Leu6, Gly8-Ser9 and Glu21-Arg22 of oxidized insulin B chain.
Article
Hydrolytic reactions between various palladium(II) complexes of the type cis-[Pd(L)(H2O)2]2+ in which L is a chelating diamine (ethylenediamine, en; 1,2-propylenediamine, 1,2-pn; N-methylethylenediamine, Meen; isobutylenediamine, ibn and N,N,N′,N′-tetramethylethylenediamine, Me4en) or S,N-coordinated amino acid (S-methyl l-cysteine, MeS-l-HCys and l-methionine, l-HMet) and N-acetylated l-histidylglycine (MeCO-His-Gly), were studied by 1H NMR spectroscopy. The reactions were carried out in the pH range 2.0–2.5 and at 60°C. In all these reactions, a palladium(II) complex bound to a histidine residue effects the regioselective cleavage of the amide bond involving the carboxylic group of histidine. We found that the rate of hydrolysis decreases as the steric bulk of the palladium(II) complex increases (en>1,2-pn>Meen>MeS-l-HCys>ibn>L-HMet>Me4en). The observed rates of hydrolytic reaction are discussed in terms of steric hindrance of the chelating diamine or sulfur-containing amino acid on the palladium(II) complexes. This study is an important step in the development of new palladium(II) complexes as artificial metallopeptidases.
Article
Electrochemical and EPR studies show that the dioxygen-induced decarboxylation and hydroxylation of [Ni-II(GGH-H--2)](-), where GGH is glycyl-glycyl-L-histidine (HL), in aqueous solution occurs via a Ni-III intermediate; the product [Ni-II(Gly-Gly-alpha-hydroxy-D,L-histamine-H-2)].3H(2)O is shown by X-ray crystallography to contain square-planar Ni-II coordinated to the terminal amino group [Ni-N, 1:932(3) Angstrom], two deprotonated amide N's [1.884(3) and 1.831(3) Angstrom] and imidazole delta N [1.908(3) Angstrom].
Article
Detailed kinetic studies on the hydrolysis of glycylserine (Gly-Ser) and glycylglycine (Gly-Gly) in the presence of the dimeric zirconium(iv)-substituted Keggin type polyoxometalate (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]·7H2O () were performed by a combination of (1)H, (13)C and (31)P NMR spectroscopy. The observed rate constants for the hydrolysis of Gly-Ser and Gly-Gly at pD 5.4 and 60 °C were 63.3 × 10(-7) s(-1) and 4.44 × 10(-7) s(-1) respectively, representing a significant acceleration as compared to the uncatalyzed reactions. The pD dependence of the rate constant for both reactions exhibited a bell-shaped profile with the fastest hydrolysis observed in the pD range of 5.5-6.0. Interaction of with Gly-Ser and Gly-Gly via their amine nitrogen and amide oxygen was proven by (13)C NMR spectroscopy. The effective hydrolysis of Gly-Ser in the presence of is most likely a combination of the polarization of the amide oxygen due to its binding to the Zr(iv) ion in and the intramolecular attack of the Ser hydroxyl group on the amide carbonyl carbon. The effect of temperature, inhibitors, and ionic strength on the hydrolysis rate constant was also examined. The solution structure of was investigated by means of (31)P NMR spectroscopy, revealing that its stability is highly dependent on pH, concentration and temperature. A 2.0 mM solution of was found to be fully stable under hydrolytic conditions (pD 5.4 and 60 °C) both in the presence and in the absence of the dipeptides.