Peptide Carbonyl Research Articles

Mechanism of Protease Resistance of D-Amino Acid Residue Containing Cationic Antimicrobial Heptapeptides.

Antimicrobial peptides (AMPs) have been an alternate promising class of therapeutics in combating global antibiotic resistance threat. However, the short half-life of AMPs, owing to protease degradability, is one of the major bottlenecks in its commercial success. In this study, we have developed all-D-amino acid containing small cationic peptides P4C and P5C, which are completely protease-resistant, noncytotoxic, nonhemolytic, and potent against the ESKAPE pathogens in comparison to their L analogues. MD simulations suggested marginal improvement in the peptide-binding affinity to the membrane-mimetic SDS micelle (∼ 1 kcal/mol) in response to L → D conversion, corroborating the marginal improvement in the antimicrobial activity. However, L → D chirality conversion severely compromised the peptide:protease (trypsin) binding affinity (≥10 kcal/mol). The relative distance between the scissile peptide carbonyl and the catalytic triad of the protease (H57, D102, and S195) was found to be significantly altered in the D-peptide:protease complex (inactive conformation) relative to the active L-peptide:protease complex. Thus, the poor binding affinity between D-peptides and the protease, resulting in the inactive complex formation, explained their experimentally observed proteolytic stability. This mechanistic insight might be extended to the proteolytic stability of the D-peptides in general and stimulate the rational design of protease-resistant AMPs.

Unraveling the interaction of bisphenol A with collagen and its effect on conformational and thermal stability

Evidence suggests the association of bisphenol A (BPA) with increased collagen (COL) expression in the development of fibrosis. Ultraviolet and fluorescence spectra on collagen-BPA interaction showed that 100 ng/ml of BPA initiated loosening of protein backbone through unfolding with exposure of tyrosine residues resulting in an intermediate “Molten Globule” state, which later aggregated with 1 μg/ml of BPA indicated with an apparent red-shift. Conformational changes with CD and ATR-FTIR showed disappearance of negative band with broadening and shifting of peptide carbonyl groups. Light scattering findings with TEM images presented initial dissolution followed by unordered thick fibrillar bundles with 30 μg/ml BPA. The complex was pH sensitive, with calorimetric thermogram revealing increased thermal stability requiring 83°C to denature. Hydrogen bonds of 2.8 Å with hydrophobic interactions of BPA in all grooves of collagen molecule with same pattern and binding energy (−4.1 to −3.9 kcal/mol) confirmed the intensity of aggregate formation via in-silico docking.

Visualizing Tetrahedral Oxyanion Bound in HIV-1 Protease Using Neutrons: Implications for the Catalytic Mechanism and Drug Design

HIV-1 protease is indispensable for virus propagation and an important therapeutic target for antiviral inhibitors to treat AIDS. As such inhibitors are transition-state mimics, a detailed understanding of the enzyme mechanism is crucial for the development of better anti-HIV drugs. Here, we used room-temperature joint X-ray/neutron crystallography to directly visualize hydrogen atoms and map hydrogen bonding interactions in a protease complex with peptidomimetic inhibitor KVS-1 containing a reactive nonhydrolyzable ketomethylene isostere, which, upon reacting with the catalytic water molecule, is converted into a tetrahedral intermediate state, KVS-1TI. We unambiguously determined that the resulting tetrahedral intermediate is an oxyanion, rather than the gem-diol, and both catalytic aspartic acid residues are protonated. The oxyanion tetrahedral intermediate appears to be unstable, even though the negative charge on the oxyanion is delocalized through a strong n → π* hyperconjugative interaction into the nearby peptidic carbonyl group of the inhibitor. To better understand the influence of the ketomethylene isostere as a protease inhibitor, we have also examined the protease structure and binding affinity with keto-darunavir (keto-DRV), which similar to KVS-1 includes the ketomethylene isostere. We show that keto-DRV is a significantly less potent protease inhibitor than DRV. These findings shed light on the reaction mechanism of peptide hydrolysis catalyzed by HIV-1 protease and provide valuable insights into further improvements in the design of protease inhibitors.

Amyloid from a histochemical perspective. A review of the structure, properties and types of amyloid, and a proposed staining mechanism for Congo red staining

ABSTRACT Amyloid is a diverse group of unrelated peptides or proteins that have positive functionality or are associated with various pathologies. Despite vast differences, all amyloids share several features that together uniquely define the group. 1) All amyloids possess a characteristic cross-ß pattern with X-ray diffraction typical of ß-sheet secondary protein structures. 2) All amyloids are birefringent and dichroic under polarizing microscopy after staining with Congo red, which indicates a crystalline-like (ordered) structure. 3) All amyloids cause a spectral shift in the peak wavelength of Congo red with conventional light microscopy due to perturbation of π electrons of the dye. 4) All amyloids show heightened intensity of fluorescence with Congo red, which suggests an unusual degree of packing of the dye onto the substrate. The ß portion of amyloid molecules, the only logical substrate for specific Congo red staining under histochemical conditions, consists of a stack of ß-sheets laminated by hydrophilic and hydrophobic interactions between adjacent pairs. Only the first and last ß-sheets are accessible to dyes. Each sheet is composed of numerous identical peptides running across the width of the sheet and arranged in parallel with side chains in register over the length of the fibril. Two sets of grooves are bordered by side chains. X grooves run perpendicular to the long axis of the fibril; these grooves are short (the width of the sheet) and number in the hundreds or thousands. Y grooves are parallel with the long axis. Each groove runs the entire length of the fibril, but there are very few of them. While Congo red is capable of ionic bonding with proteins via two sulfonic acid groups, physical constraints on the staining solution preclude ionic interactions. Hydrogen bonding between dye amine groups and peptide carbonyls is the most likely primary bonding mechanism, because all ß-sheets possess backbone carbonyls. Various amino acid residues may form secondary bonds to the dye via any of three van der Waals forces. It is possible that Congo red binds within the Y grooves, but that would not produce the characteristic staining features that are the diagnostic hallmarks of amyloid. Binding in the X grooves would produce a tightly packed series of dye molecules over the entire length of the fibril. This would account for the signature staining of amyloid by Congo red: dichroic birefringence, enhanced intensity of fluorescence and a shift in visible absorption wavelength.

Engineering Proton Transfer in Photosynthetic Oxygen Evolution: Chloride, Nitrate, and Trehalose Reorganize a Hydrogen-Bonding Network.

Photosystem II oxidizes water at a Mn4CaO5 cluster. Oxygen evolution is accompanied by proton release through a 35 Å hydrogen-bonding network to the lumen. The mechanism of this proton-transfer reaction is not known, but the reaction is dependent on chloride. Here, vibrational spectroscopy defines the functional properties of the proton-transfer network using chloride, bromide, and nitrate as perturbative agents. As assessed by peptide C═O frequencies, bromide substitution yields a spectral Stark shift because of its increase in ionic radius. Nitrate substitution leads to more complex spectral changes, consistent with an overall increase in hydrogen-bonding interactions with the peptide backbone. The effects are similar to spectral changes previously documented in site-directed mutations in a putative lumenal pathway. Importantly, the effects of nitrate are reversed by the osmolyte, trehalose. Trehalose is known to alter hydrogen-bonding interactions in proteins. Trehalose addition also reverses a shift in an internal hydronium ion signal, consistent with an alteration in its p Ka value and a change in the basicity of bound nitrate. The spectra provide evidence that the proton-transfer pathway contains peptide carbonyl groups, internal water, a hydronium ion, and amino acid side chains. These experiments also show that the proton-transfer pathway functionally adapts to changes in electric field, p Ka, and hydrogen bonding and thereby optimizes proton transfer to the lumen.

Hydrogen-bonding network at the cytoplasmic region of a light-driven sodium pump rhodopsin KR2

Light-driven sodium-pumping rhodopsins are able to actively transport sodium ions. Structure/function studies of Krokinobacter eikastus rhodopsin 2 (KR2) identified N61 and G263 at the cytoplasmic surface constituting the “Ion-selectivity filter” for sodium ions, while retinal Schiff base acts as the light “Switch and Gate” for transport of sodium ions. Q123 is located between the two regions, and plays an important role for the pump function, which was implicated by functional, spectroscopic, X-ray crystallographic and computational studies. According to the atomic structure of KR2, Q123 is involved in the hydrogen-bonding network at the cytoplasmic region, together with S64, protein-bound waters, and peptide carbonyl of K255 bound to the chromophore. To gain the detailed structural information around Q123, here we compared light-induced difference Fourier-transform infrared (FTIR) spectra at 77 K between the wild-type (WT) and mutant proteins of KR2, such as Q123A, Q123V, and S64A. The obtained spectra were very similar between WT and these mutants, whereas the observed mutation effects enabled us to identify vibrations of the hydrogen-bonding network at the Q123 and S64 region. This is unique for KR2, not for the corresponding mutations in a light-driven proton-pump bacteriorhodopsin (BR). Hydrogen-bonding alteration is absent for the mutants of KR2, suggesting that proper inter-helical connectivity of helices B, C, and G is important for protein structural changes for sodium-pump function, which is controlled by the region around Q123.

Biosynthesis of Translation Inhibitor Klebsazolicin Proceeds through Heterocyclization and N-Terminal Amidine Formation Catalyzed by a Single YcaO Enzyme.

Klebsazolicin (KLB) is a recently discovered Klebsiella pneumonia peptide antibiotic targeting the exit tunnel of bacterial ribosome. KLB contains an N-terminal amidine ring and four azole heterocycles installed into a ribosomally synthesized precursor by dedicated maturation machinery. Using an in vitro system for KLB production, we show that the YcaO-domain KlpD maturation enzyme is a bifunctional cyclodehydratase required for the formation of both the core heterocycles and the N-terminal amidine ring. We further demonstrate that the amidine ring is formed concomitantly with proteolytic cleavage of azole-containing pro-KLB by a cellular protease TldD/E. Members of the YcaO family are diverse enzymes known to activate peptide carbonyls during natural product biosynthesis leading to the formation of azoline, macroamidine, and thioamide moieties. The ability of KlpD to simultaneously perform two distinct types of modifications is unprecedented for known YcaO proteins. The versatility of KlpD opens up possibilities for rational introduction of modifications into various peptide backbones.

Gas-phase complexes of Ni2+ and Ca2+ with deprotonated histidylhistidine (HisHis): A model case for polyhistidyl-metal binding motifs

In the complex formed between the calcium cation (Ca2+) and a deprotonated HisHis dipeptide, the complex adopts a charge solvation (CS) structure. Ca2+, a weak binding main group metal cation, interacts with the oxygens of the peptide carbonyl moiety and the deprotonated C-terminus. In contrast, the much stronger binding Ni2+ cation deprotonates the peptide nitrogen and induces an iminolate (Im) ligand structure in the [Ni(HisHis-H)]+ complex ion. The combination of infrared multiple-photon dissociation (IRMPD) spectroscopy and quantum chemistry evidence these two representative binding motifs. The iminolate coordination pattern identified and characterized in the [Ni(HisHis-H)]+ complex serves as a model case for nickel complexes of poly-histidyl-domains and is thereby also of interest to better understand the fundamentals of immobilized metal ion affinity chromatography as well as of Ni co-factor chemistry in enzymology.

Structural Analysis of Hand Drawn Bumblebee Bombus terrestris Silk.

Bombus terrestris, commonly known as the buff-tailed bumblebee, is native to Europe, parts of Africa and Asia. It is commercially bred for use as a pollinator of greenhouse crops. Larvae pupate within a silken cocoon that they construct from proteins produced in modified salivary glands. The amino acid composition and protein structure of hand drawn B. terrestris, silk fibres was investigated through the use of micro-Raman spectroscopy. Spectra were obtained from single fibres drawn from the larvae salivary gland at a rate of 0.14 cm/s. Raman spectroscopy enabled the identification of poly(alanine), poly(alanine-glycine), phenylalanine, tryptophan, and methionine, which is consistent with the results of amino acid analysis. The dominant protein conformation was found to be coiled coil (73%) while the β-sheet content of 10% is, as expected, lower than those reported for hornets and ants. Polarized Raman spectra revealed that the coiled coils were highly aligned along the fibre axis while the β-sheet and random coil components had their peptide carbonyl groups roughly perpendicular to the fibre axis. The protein orientation distribution is compared to those of other natural and recombinant silks. A structural model for the B. terrestris silk fibre is proposed based on these results.

Ion-Protein Interactions between a Potassium Channel and Alkali Metal Cations Studied by ATR-FTIR Spectroscopy

Potassium channels are essential for selective permeation of potassium ions in cell membrane. The selectivity is achieved by the specific part called “selectivity filter”, which is composed of several carbonyl groups centripetally toward the center of the pore. X-ray crystallography clearly showed that each potassium ion is favorably coordinated by eight carbonyl oxygens in the selectivity filters of various kinds of potassium channels. Further analysis was performed on a well-known potassium channel, KcsA, where the coordination manners of alkali metal cations in the selectivity filter are somewhat different from each other. In particular, sodium ion is coordinated by four carbonyl groups and the structure of the filter is deformed in the center of the filter. To understand the ion-protein interactions of potassium channels in more detail, the structure around the selectivity filter should be analyzed by other physicochemical methods, such as infrared difference spectroscopy.The infrared difference spectroscopy on KcsA with ion-exchange reaction was firstly reported in 2012 [1]. The significant spectral change was observed in the amide I mode of the peptide carbonyl groups, which were assigned to the selectivity filter and nearby pore helices by mutation [1] and a computational method [2]. The interactions with alkali metal cations were further investigated [3], revealing that the selectivity filter carbonyls coordinating Rb+ or Cs+ adopt a conformation similar to those coordinating K+, while those coordinating Li+ or Na+ considerably differ from those coordinating K+. Similar approach applied on a mammalian potassium channel is now in progress.[1] Y. Furutani et al. J. Phys. Chem. Lett. 3, 3806-3810, 2012[2] P. Stevenson et al. J. Phys. Chem. B 119, 5824-5831, 2015[3] Y. Furutani et al. Biophysics and Physicobiology 12, 37-45, 2015

Dynamic Water-Mediated Hydrogen Bonding in a Collagen Model Peptide.

In the canonical (G-X-Y)(n) sequence of the fibrillar collagen triple helix, stabilizing direct interchain hydrogen bonding connects neighboring chains. Mutations of G can disrupt these interactions and are linked to connective tissue diseases. Here we integrate computational approaches with nuclear magnetic resonance (NMR) to obtain a dynamic view of hydrogen bonding distributions in the (POG)(4)(-)(POA)-(POG)(5) peptide, showing that the solution conformation, dynamics, and hydrogen bonding deviate from the reported X-ray crystal structure in many aspects. The simulations and NMR data provide clear evidence of inequivalent environments in the three chains. Molecular dynamics (MD) simulations indicate direct interchain hydrogen bonds in the leading chain, water bridges in the middle chain, and nonbridging waters in the trailing chain at the G → A substitution site. Theoretical calculations of NMR chemical shifts using a quantum fragmentation procedure can account for the unusual downfield NMR chemical shifts at the substitution sites and are used to assign the resonances to the individual chains. The NMR and MD data highlight the sensitivity of amide shifts to changes in the acceptor group from peptide carbonyls to water. The results are used to interpret solution NMR data for a variety of glycine substitutions and other sequence triplet interruptions to provide new connections between collagen sequences, their associated structures, dynamical behavior, and their ability to recognize collagen receptors.

Luminescence due to peptide linkage observed in L-cysteine molecules irradiated by infrared laser light

The sequence of amino acids in peptide chains consisting of proteins is the most fundamental information of living things. A direct and nondestructive method of reading is highly required as an alternative to the method based on the gene analysis. Luminescence detection is a very sensitive tool for investigating various materials. In order to find characteristic luminescence of each amino acid we study L-cysteine and L-tyrosine using UV laser of 3.36eV with pulse duration of 1.5ps. In addition to a common 2.66eV band of the luminescence we have found 2.89eV band for L-cysteine and 2.92eV band for L-tyrosine. It can be interpreted that the side chain makes difference on the luminescence by affecting the peptide linkage or carbonyl group.

Heterogeneity of peptide adducts with carbonylated lipid peroxidation products.

Highly reactive lipid peroxidation-derived carbonyls (oxoLPP) modify protein nucleophiles via Michael addition or Schiff base formation. Once formed, Michael adducts can be further stabilized via cyclic hemiacetals with or without loss of water. Depending on the mechanism of their formation, peptide-oxoLPP can carry aldehyde or keto groups and thus be a part of the total protein carbonylation level. If a carbonyl function is lost during consecutive reactions, the oxoLPP-peptide adducts will not be detected using the common carbonyl labeling protocols. Because of the differences in adduct stabilities, it is possible to address the heterogeneity of peptide/protein-oxoLPP adducts by careful evaluation of tandem mass spectra of modified peptides. Here, we used hydrophilic interaction liquid chromatography-tandem mass spectrometry analysis of lysine, cysteine and histidine containing model peptides co-incubated with oxidized 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine to characterize the collision-induced dissociation behavior of peptide-carbonyl adducts. Numerous modifications were detected based on the analysis of tandem mass spectra, including Schiff bases on lysine (two), Michael adducts on lysine (six), cysteine (eleven) and histidine (two), as well as 4-hydroxy-2-aldehydes derived dehydrated cyclic hemiacetals on cysteine (five) and histidine (one). Additionally, cysteine and histidine side chains were modified by lipid-bound aldehydes as Michael adducts and dehydrated hemiacetals. The tandem mass spectra revealed collision-induced dissociation characteristics specific for each class of oxoLPP-peptide adducts.

Insights into the oxygen-based ligand of the low pH component of the Cu(2+)-amyloid-β complex.

In spite of significant experimental effort dedicated to the study of Cu(2+) binding to the amyloid beta (Aβ) peptide, involved in Alzheimer's disease, the nature of the oxygen-based ligand in the low pH component of the Cu(2+)-Aβ(1-16) complex is still under debate. This study reports density-functional-theory-based calculations that explore the potential energy surface of Cu(2+) complexes including N and O ligands at the N-terminus of the Aβ peptide, with a focus on evaluating the role of Asp1 carboxylate in copper coordination. Model conformers including 3, 6, and 17 amino acids have been used to systematically study several aspects of the Cu(2+)-coordination such as the Asp1 side chain conformation, local peptide backbone geometry, electrostatic and/or hydrogen bond interactions, and number and availability of Cu(2+) ligands. Our results show that the Asp1 peptide carbonyl binds to Cu(2+) only if the coordination number is less than four. In contrast, if four ligands are available, the most stable structures include the Asp1 carboxylate in equatorial position instead of the Asp1 carbonyl group. The two lowest energy Cu(2+)-Aβ(1-17) models involve Asp1 COO(-), the N-terminus, and His6 and His14 as equatorial ligands, with either a carbonyl or a water molecule in the axial position. These models are in good agreement with experimental data reported for component I of the Cu(2+)-Aβ(1-16) complex, including EXAFS- and X-ray-derived Cu(2+)-ligand distances, Cu(2+) EPR parameters, and (14)N and (13)C superhyperfine couplings. Our results suggest that at low pH, Cu(2+)-Aβ species with Asp1 carboxylate equatorial coordination coexist with species coordinating the Asp1 carbonyl. Understanding the bonding mechanism in these species is relevant to gain a deeper insight on the molecular processes involving copper-amyloid-β complexes, such as aggregation and redox activity.

Polarized Raman Spectroscopy of Aligned Insulin Fibrils.

Amyloid fibrils are associated with many neurodegenerative diseases. The application of conventional techniques of structural biology, X-ray crystallography and solution NMR, for fibril characterization is limited because of the non-crystalline and insoluble nature of the fibrils. Here, polarized Raman spectroscopy was used to determine the orientation of selected chemical groups in aligned insulin fibrils, specifically of peptide carbonyls. The methodology is solely based on the measurement of the change in Raman scattered intensity as a function of the angle between the incident laser polarization and the aligned fibrils. The order parameters 〈 P2 〉 and 〈 P4 〉 of the orientation distribution function were obtained, and the most probable distribution of C=O group orientation was calculated. The results indicate that the peptides' carbonyl groups are oriented at an angle of 13±5° from the fibril axis, which is in consistent with previously reported qualitative descriptions of an almost parallel orientation of the C=O groups relative to the main fibril axis.

Water scaffolding in collagen: Implications on protein dynamics as revealed by solid‐state NMR

Solid-state NMR studies of collagen samples of various origins confirm that the amplitude of collagen backbone and sidechain motions increases significantly on increasing the water content. This conclusion is supported by the changes observed in three different NMR observables: (i) the linewidth dependence on the 1H decoupling frequency; (ii) 13C CSA changes for the peptide carbonyl groups, and (iii) dephasing rates of 1H-13C dipolar couplings. In particular, a nearly threefold increase in motional amplitudes of the backbone librations about C-Cα or N-Cα bonds was found on increasing the added water content up to 47 wt%D2 O. On the basis of the frequencies of NMR observables involved, the timescale of the protein motions dependent on the added water content is estimated to be of the order of microseconds. This estimate agrees with that from wideline T2(1)H NMR measurements. Also, our wideline 1H NMR measurements revealed that the timescale of the microsecond motions in proteins reduces significantly on increasing the added water content, i.e., an ∼15-fold increase in protein motional frequencies is observed on increasing the added water content to 45 wt% D2 O. The observed changes in collagen dynamics is attributed to the increase in water translational diffusion on increasing the amount of added water, which leads to more frequent "bound water/free water" exchange on the protein surface, accompanied by the breakage and formation of new hydrogen bonds with polar functionalities of protein.

Expression of Some Pro-inflammatory Cytokines and Inflammatory Mediators in Experimental Model of Trichinella Spiralis

Trichinellosis is a zoonosis acquired by the ingestion of undercooked meat containing the infective larvae of Trichinella spiralis (T. spiralis). Despite veterinary public health efforts to control it, re-emergence of the disease has been observed in many areas of the world in the past 10–20 years. Although ivermectin (IVM) achieved high efficacy as larvicidal in trichinellosis, the possibility of disease transmission to other hosts is still present that will hinder the eradication of that serious disease. Therefore, our goal is to determine the efficacy of IVM on the subsequent infectivity of T. spiralis larvae in experimental animals through parasitological and biochemical studies of the intestinal phase. We found that the proinflammatory cytokine; interleukin-1β (IL-1β) and epithelial neutrophil activating peptide 78 (ENA-78) mRNA expression were decreased in group II which was infected by IVM- treated T. spiralis larvae on the 5th day post infection, also the same group showed the lowest levels of vasoactive intestinal peptide (VIP) and protein carbonyl (PCO) as well as the lowest activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) enzymes. The lowest adult and larval counts were observed in group II at the 5 th day post infection with highly significant differences in comparison to other groups. In conclusion, IVM appears to affect the infectivity of T. spiralis larvae with subsequent decrease of T. spiralis adults and total larval count. These effects were more pronounced when IVM was given early in the infection. IVM is feasible and useful drug in the treatment of current infection and control of subsequent infections with T. spiralis.

Calcium and the Hydrogen-Bonded Water Network in the Photosynthetic Oxygen-Evolving Complex.

In photosynthesis, photosystem II evolves oxygen from water at a Mn4CaO5 cluster (OEC). Calcium is required for biological oxygen evolution. In the OEC, a water network, extending from the calcium to four peptide carbonyl groups, has recently been predicted by a high-resolution crystal structure. Here, we use carbonyl vibrational frequencies as reporters of electrostatic changes to test the presence of this water network. A single flash, oxidizing Mn(III) to Mn(IV) (the S1 to S2 transition), upshifted the frequencies of peptide C═O bands. The spectral change was attributable to a decrease in C═O hydrogen bonding. Strontium, which supports a lower level of steady state activity, also led to an oxidation-induced shift in C═O frequencies, but treatment with barium and magnesium, which do not support activity, did not. This work provides evidence that calcium maintains an electrostatically responsive water network in the OEC and shows that OEC peptide carbonyl groups can be used as solvatochromic markers.

The New Serine Protease Song (to the tune of Rudolf the Red‐nosed Reindeer)

All serine proteases Work almost identically Using amino acid Triads catalytically First they bind peptide substrates Holding onto them so tight Changing their structure when they Get them in the S1 site Then there are electron shifts At the active site Serine gives up its proton As the RE-ac-tion goes on Next the alkoxide ion Being so electron rich Grabs peptide's carbonyl group Breaks its bond without a hitch So one piece is bound to it The other gets set free Water has to act next to Let the final fragment loose Then it's back where it started Waiting for a peptide chain That it can bind itself to Go and start all o'er again Additional Supporting Information (an .mp3 file of this song) may befound in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Amino acid side chain induced selectivity in the hydrolysis of peptides catalyzed by a Zr(iv)-substituted Wells–Dawson type polyoxometalate

In this paper the reactivity of K15H[Zr(α2-P2W17O61)2]·25H2O (1), 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.