Molecular dynamics simulations of Cu(II) and the PHGGGWGQ octapeptide

The interaction between Cu2+ and the copper-binding octapeptide region in the human prion protein has been investigated by molecular dynamics simulations. In total four different nonbonded and bonded models were used in the study. Charge sets containing atomic partial charges were developed for these models. Out of the considered models, the bonded model performed physically in the most correct way. The simulations with the bonded model showed that the water molecules in the axial position are very labile. The tryptophan indole ring can remain in a stable position on top of the equatorial coordination plane of copper without water mediation. Strong aromatic interaction was observed between the imidazole and indole rings. The nonbonded models showed a tendency for water-mediated interaction between the copper ion and different carbonyl oxygen atoms. In the case of the bonded model, a carbonyl group could also interact directly with the copper ion in one of the apical position.     

Biological fingerprinting analysis of the interactome of a kinase inhibitor in human plasma by a chemiproteomic approach

In this study, a gel free chemiproteomic method based on chromatography was developed and applied for the biological fingerprinting analysis of complex biological system. p-Aminobenzamidine (ABA), an inhibitor of trypsin-like serine proteases, was immobilized for characterizing their interacting proteins in human plasma. By the proteomic analysis method, 214 proteins were identified with obvious affinity to the immobilized ABA. By searching the sequences of above proteins with consensus patterns of the two active sites, seven proteins belong to trypsin-like serine protease group were found. Based on the Gene Ontology annotation, the identified trypsin-like serine proteases have the function of catalytic activity and calcium ion binding, and are mainly involved in the biological process of blood coagulation. Eight more other proteins related to calcium ion binding and blood coagulation were found. Nearly all of these proteins cannot be identified by directly analyzing the plasma sample demonstrating the chemiproteomics a useful approach to characterize interacting proteins in the low abundance range.     

Surfactant systems as enzyme models

The large catalyses induced by enymes in living systems have prompted interest in in vitro reproduction of the rate enhancements and specificities observed, with a view either to elucidating the mechanism of the enyme catalyzed reaction or to making use of the catalysis in some non-living process.Characterization and utilization of surfactant systems as models for serine proteases are the main feature of this review. These enzymes, in which the serine residue becomes acylated within the active site during the reaction, serve in the hydrolysis of carbonyl containing proteins and esters. For this reason, most workers have modelled features of the trypsin enzyme, α-chrymotrypsin, and have studied the effect on the reactivity of reactive esters or amides, of functionalized surfactants containing hydrozy-, oxime, amino- and imidazole headgroups. Recent studies have extended to thiol containing surfactants as models for the cysteine proteases. Examples are given to illustrate the reaction mechanism and products of these mimetic agents.     

Effect of a charge relay on the vibrational frequencies of carbonmonoxy iron porphine adducts: the coupling of changes in axial ligand bond strength and porphine core size.

The effect of a charge relay involving Asp-His-Fe in peroxidase enzymes is explored using density functional theory (DFT) calculations of vibrational spectra and potential energy surfaces of carbonmonoxy model systems. The series of models consists of a carbonmonoxy iron porphine molecule with a trans imidazole ligand hydrogen-bonded to six different partners at the Ndelta position. Calculations on the oxy system and on models of the Asp-His-Ser catalytic triad of serine proteases were also performed to obtain an understanding of how the redistribution of charge in these systems may contribute to enzymatic function. The goal of the study is to relate the experimental frequencies in resonance Raman and Fourier transform infrared studies to bonding that is important for the function of heme enzymes. Calculations of both axial and in-plane modes exhibit trends that agree with experimental data. Comparisons of the charge distribution on the different models show that polarization of iron carbonomonoxy bonds consistent with the mechanism for peroxidase function leads to a frequency reduction in the C-O stretching mode nuCO. The combination of axial trans sigma-bonding and pi-bonding effects that include expansion of the porphine core result in little change in the Fe-C stretching frequency nuFe-CO in the series of molecules studied with different Ndelta-H hydrogen bonding. A particular role for the core size is discussed that demonstrates the applicability of trends observed in vibrational spectroscopy of hemes to the charge relay mechanism and other axial ligation effects. The bonding interactions described account for the increase in electron density on bound diatomic ligands, which is required for peroxidase function.     

Gem-diol Generation in Copper and Zinc N-methyl-2-imidazolecarboxaldehyde Complexes: Solid-state NMR,EPR and Single-Crystal X-ray Studies

Novel coordination compounds, mono- ( 1 ) and binuclear copper complexes ( 2 ) and a zinc complex ( 3 ) were synthesized and studied through single-crystal X-ray crystallography, solid-state NMR and EPR techniques to determine the chemical functionalization of the carbonyl group in N-methyl-2-imidazolecarboxaldehyde ligand. Particularly, molecules containing carbonyl groups are versatile ligands that give rise to a wide range of new materials due to the high reactivity of the carbonyl group. However, the chemical identification of the functional group present in these coordination compounds is a challenge because the copper ion affects the NMR signals. In this sense, X-ray crystallography becomes an indispensable tool for the analysis. The imidazole ligands in copper complexes 1 and 2 were found to be the aldehyde and the gem-diol forms, respectively. Furthermore, the gem-diol and carboxylate moieties were detected in the crystal lattice for the zinc complex 3 and studied by solution- and solid-state NMR.     

Probing the configurational space of a metalloprotein core: an ab initio molecular dynamics study of Duo Ferro 1 binuclear Zn cofactor

We present three theoretical models of various degree of completeness to explore the chemical phase space available to the Glu4His2Zn2 cofactor found in the four-helix bundle of de novo designed metalloprotein Duo Ferro 1. We have found that the planewave DFT geometry optimization of 94-atom Model I, which contains both the protein scaffold constraints as well as the second shell hydrogen bonding network, reproduces the crystal structure bonding with remarkable accuracy (0.34 A). Surprisingly, the geometry optimization of 66-atom Model II (lacking the second shell hydrogen bonding) and 48-atom Model III (being also free of the protein scaffold constraints) still result in the fidelity with the crystallographic structure (RMSDs 0.29 and 0.34 A, respectively). To examine whether these structures are close to the global minimum as well as to investigate various conformational transitions to which the di-Zn cofactor may be susceptible to, we have carried out a 10 ps Car-Parrinello Molecular Dynamics (CPMD) simulation of Model III. We suggest that weak hydrogen bonds between imidazole hydrogens and carboxylate oxygens modulate the dynamical behavior of the system. One part of the molecule was found to be rigid due to the particular H(imidazole)-O(carboxylate) interaction restricting both the motion of the imidazole ring as well as the terminal carboxylate conformational mobility. The second half of the system was very flexible demonstrating a coupling of a transient formation of H(imidazole)-O(carboxylate) bonds with the spinning of the imidazole ring and syn-anti isomerization of the terminal carboxylate group. In addition, two low-energy snapshots from the 10 ps CPMD run were quenched, and their geometries were optimized, leading to two new isomers 48 kJ/mol lower in energy than the one associated with the crystal structure. We suggest that periodic quenching of the CPMD simulation snapshots of a minimalist model may be used as an efficient method to generate a large number of competitive local minima, which may be consequently pruned by imposing the protein scaffold constraints as well as further tuned by the second shell hydrogen bonding network.     

Computational investigation of irreversible inactivation of the zinc-dependent protease carboxypeptidase A

Zinc proteases are ubiquitous and the zinc ion plays a central function in the catalytic mechanism of these enzymes. A novel class of mechanism-based inhibitors takes advantage of the zinc ion chemistry in carboxypeptidase A (CPA) to promote covalent attachment of an inhibitor to the carboxylate of Glu-270, resulting in irreversible inhibition of the enzyme. The effect of the active site zinc ion on irreversible inactivation of CPA was probed by molecular orbital (MO) calculations on a series of active site models and the Cl(-) + CH(3)Cl S(N)2 reaction fragment. Point charge models representing the active site reproduced energetics from full MO calculations at 12.0 A separation between the zinc and the central carbon of the S(N)2 reaction, but at 5.0 A polarization played an important role in moderating barrier suppression. ONIOM MO/MO calculations that included the residues within 10 A of the active site zinc suggest that about 75% of the barrier suppression arises from the zinc ion and its ligands. A model of the pre-reactive complex of the 2-benzyl-3-iodopropanoate inactivator with CPA was constructed from the X-ray structure of l-phenyl lactate bound in the active site of the enzyme. The model was fully solvated and minimized by using the AMBER force field to generate the starting structure for the ONIOM QM/MM calculations. Optimization of this structure led to the barrierless S(N)2 displacement of the iodide of the inhibitor by Glu-270, assisted by interaction of the zinc ion with the leaving group. The resulting product is in good agreement with the X-ray structure of the covalently modified enzyme obtained by irreversible inhibition of CPA by 2-benzyl-3-iodopropanoate.     

Strong N−H⋅⋅⋅O Hydrogen Bonding in a Model Compound of the Catalytic Triad in Serine Proteases

Low-barrier hydrogen bond (LBHB) involvement in enzyme catalysis is examined by analysis of experimental nuclear and electron densities of a model compound for the catalytic triad in serine proteases (shown schematically), which is based on a cocrystal of betaine, imidazole, and picric acid. The three short, strong N−H⋅⋅⋅O hydrogen bonds in the structure have varying degrees of covalent bonding contributions suggesting a gradual transition to the LBHB situation.     

C-terminal amino acid residue loss for deprotonated peptide ions containing glutamic acid, aspartic acid, or serine residues at the C-terminus

Deprotonated peptides containing C-terminal glutamic acid, aspartic acid, or serine residues were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with ion production by electrospray ionization (ESI). Additional studies were performed by post source decay (PSD) in a matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometer. This work included both model peptides synthesized in our laboratory and bioactive peptides with more complex sequences. During SORI-CID and PSD, [M - H]- and [M - 2H]2- underwent an unusual cleavage corresponding to the elimination of the C-terminal residue. Two mechanisms are proposed to occur. They involve nucleophilic attack on the carbonyl carbon of the adjacent residue by either the carboxylate group of the C-terminus or the side chain carboxylate group of C-terminal glutamic acid and aspartic acid residues. To confirm the proposed mechanisms, AAAAAD was labelled by 18O specifically on the side chain of the aspartic acid residue. For peptides that contain multiple C-terminal glutamic acid residues, each of these residues can be sequentially eliminated from the deprotonated ions; a driving force may be the formation of a very stable pyroglutamatic acid neutral. For peptides with multiple aspartic acid residues at the C-terminus, aspartic acid residue loss is not sequential. For peptides with multiple serine residues at the C-terminus, C-terminal residue loss is sequential; however, abundant loss of other neutral molecules also occurs. In addition, the presence of basic residues (arginine or lysine) in the sequence has no effect on C-terminal residue elimination in the negative ion mode.     

Influence of environment on proton-transfer mechanisms in model triads from theoretical calculations

We have carried out theoretical calculations to analyze molecular interactions and proton transfer mechanisms in the formate–imidazole–water system, which may be considered the simplest model of catalytic triads in serine proteases. Computations were carried out at the density functional theory level. The effect of a dielectric environment on energy surfaces is considered using a polarizable continuum model and the self-consistent reaction field approach. The role played by inertial and noninertial polarization of this environment is emphasized. Nonequilibrium solvation effects have been estimated. The results show that there are different reaction mechanisms, concerted or stepwise, that may be competitive, depending on the nature of the molecular environment. © 1998 John Wiley & Sons, Inc. J Comput Chem 19: 1675–1688, 1998     

Spectroscopic and computational evaluation of the structure of the high-spin Fe(IV)-oxo intermediates in taurine: alpha-ketoglutarate dioxygenase from Escherichia coli and its His99Ala ligand variant

The Fe(II)- and alpha-ketoglutarate (alphaKG)-dependent dioxygenases activate O2 for cleavage of unactivated C-H bonds in their substrates. The key intermediate that abstracts hydrogen in the reaction of taurine:alphaKG dioxygenase (TauD), a member of this enzyme family, was recently characterized. The intermediate, denoted J, was shown to contain an iron(IV)-oxo unit. Other important structural features of J, such as the number, identity, and disposition of ligands in the Fe(IV) coordination sphere, are not yet understood. To probe these important structural features, a series of models for J with the Fe(IV) ion coordinated by the expected two imidazole (from His99 and His255), two carboxylate (succinate and Asp101), and oxo ligands have been generated by density functional theory (DFT) calculations, and spectroscopic parameters (M?ssbauer isomer shift, quadrupole splitting, and asymmetry parameter, 57Fe hyperfine coupling tensor, and zero field splitting parameters, D and E/D) have been calculated for each model. The calculated parameters of distorted octahedral models for J, in which one of the carboxylates serves as a monodentate ligand and the other as a bidentate ligand, and a trigonal bipyramidal model, in which both carboxylates serve as monodentate ligands, agree well with the experimental parameters, whereas the calculated parameters of a square pyramidal model, in which the oxo ligand is in the equatorial plane, are inconsistent with the data. Similar analysis of the Fe(IV) complex generated in the variant protein with His99, the residue that contributes the imidazole ligand cis to the oxo group, replaced by alanine suggests that the deleted imidazole is replaced by a water ligand. This work lends credence to the idea that the combination of M?ssbauer spectroscopy and DFT calculations can provide detailed structural information for reactive intermediates in the catalytic cycles of iron enzymes.     

The influence of structural features on facile McLafferty-type, even-electron rearrangements in tandem mass spectra of carboxylate anions

In contrast to the well-described McLafferty rearrangement in odd-electron cations, relatively little has been reported on comparable rearrangements in even-electron ions, especially negative ions. This work reports a systematic study using tandem mass spectrometry (MS/MS) fragment ion spectra of carboxylate anions having a suitably acidic proton in the gamma position. The rearrangement process was studied in both ion trap and triple quadrupole mass spectrometers; characteristic enolate anions and stable neutral products were formed at low collision energies. The process has diagnostic and analytical potential in, for example, the analysis of peptides having C-terminal serine residues and of 3-hydroxy- or 3-aminocarboxylic acids in complex mixtures.     

Spectroscopic analysis of L-histidine adsorbed on gold and silver nanoparticle surfaces investigated by surface-enhanced Raman scattering

The adsorption of l-histidine on gold (Au) and silver (Ag) nanoparticle surfaces has been comparatively analyzed by means of surface-enhanced Raman scattering (SERS). The SERS spectra of l-histidine on Ag were found to be quite different from those on Au, indicating dissimilar adsorption structures depending on metal substrates. Most peaks of l-histidine on Ag appeared to be due to coordination via the carboxylate (COO(-)) group with an imidazole ring of fairly upright geometry, whereas on Au it was assumed to adsorb with a rather flat geometry. A density functional theory (DFT) calculation was performed at the level of B3LYP/LANL2DZ to estimate the energetic stability of the binding of the imidazole ring and the carboxylate group of l-histidine with the Ag and Au atoms, respectively. Based on the DFT calculation, the carboxylate group of l-histidine was predicted to bind more favorably to Ag than to Au, and this was in line with our SERS spectral analysis.     

Synthesis and spectral studies of mixed copper(II) dipeptide complexes of imidazole and related nitrogen donors

Eight mixed copper(II) complexes of the type [Cu(II)(D)(HL)], where D = anion of glycylglycine, glycyl-L-tyrosine or glycyl-L-phenylalanine, and HL = imidazole, 1-methylimidazole, 2-methylimidazole or benzimidazole have been prepared and characterised. The visible and EPR spectral studies of these complexes indicate that they are monomeric having five coordinate square pyramidal geometry (possibly distorted) about Cu(II). The dipeptide behaves as terdentate ligand in these complexes with amino, ionised amide nitrogen and carboxylate oxygen donor atoms approximately tetragonally disposed about Cu(II). The magnetic and bonding parameters obtained by detailed EPR spectral analysis coupled with electronic absorption spectral data suggest that imidazole, 1-methylimidazole, 2-methylimidazole or benzimidazole occupies the fourth position in the tetragonal plane and water molecule occupies an axial position about Cu(II) in solid state and in solution.     

Nanomolar inhibitors of AmpC beta-lactamase

beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics, such as the penicillins and the cephalosporins. In an effort to combat these enzymes, a combination of stereoselective organic synthesis, enzymology, microbiology, and X-ray crystallography was used to design and evaluate new carboxyphenyl-glycylboronic acid transition-state analogue inhibitors of the class C beta-lactamase AmpC. The new compounds improve inhibition by over 2 orders of magnitude compared to analogous glycylboronic acids, with K(i) values as low as 1 nM. On the basis of the differential binding of different analogues, the introduced carboxylate alone contributes about 2.1 kcal/mol in affinity. This carboxylate corresponds to the ubiquitous C3(4)' carboxylate of beta-lactams, and this energy represents the first thermodynamic measurement of the importance of this group in molecular recognition by class C beta-lactamases. The structures of AmpC in complex with two of these inhibitors were determined by X-ray crystallography at 1.72 and 1.83 A resolution. These structures suggest a structural basis for the high affinity of the new compounds and provide templates for further design. The highest affinity inhibitor was 5 orders of magnitude more selective for AmpC than for characteristic serine proteases, such as chymotrypsin. This inhibitor reversed the resistance of clinical pathogens to the third generation cephalosporin ceftazidime; it may serve as a lead compound for drug discovery to combat bacterial resistance to beta-lactam antibiotics.     

Synthesis and Characterization of Iron(III) Complexes of 5‐(8‐Carboxy‐1‐naphthyl)‐10, 15, 20‐tritolyl Porphyrin

5‐(8‐Carboxy‐1‐naphthyl)‐10, 5, 20‐tritolyl porphyrin (H₃CNTTP) and its iron(III) complexes, [Fe(CNTTP)]₂ and [Fe(CNTTP)(N‐MeIm)₂], were synthesized and characterized. X‐ray crystallography revealed that the carboxylate group is “hanging” over the porphyrin plane. The rigid framework makes the distance between the carboxylate oxygen and iron in the same porphyrin too long to form a coordination bond. On the other hand, the carboxylate group is not bulky enough to block the axial binding site. In the presence of OH^–, the carboxylate oxygen is coordinated to iron in the symmetry‐related unit, which led to the dimeric structure, [Fe(CNTTP)]₂. In the presence of excess N‐methylimidazole, a six‐coordinate species, [Fe(CNTTP)(N‐MeIm)₂], was obtained. In such a structure, CH ··· O interactions between the carboxylate group and imidazole probably play an important role to determine the orientation of imidazole plane. Two imidazole planes have relative parallel orientation. For [Fe(CNTTP)(N‐MeIm)₂], ¹H NMR shows pyrrole protons at the region –10 to –25 ppm. EPR shows rhombic spectrum. Those suggest [Fe(CNTTP)(N‐MeIm)₂] is a type II low‐spin iron(III) porphyrinate.     

Surface acidity and basicity of functionalized silica particles

Tetraethoxysilane has been co-hydrolyzed with functionalized organosilanes in a modified Stöber process to produce silica particles with amino, carboxylate or dihydroimidazole groups on the surface. The effects of reaction conditions and the loading of the functionalized organosilane on particle size was examined by TEM. Fluorescence spectroscopy of the surface amino groups covalently modified with fluorescamine, and the surface carboxylate groups with 4-bromomethyl-6,7-dimethoxycoumarin, demonstrated that these functional groups were accessible for further reaction. Changes in surface acidity and basicity caused by the presence of functional groups (amine, dihydroimidazole, carboxylate) on the particle surface were determined using an indicator titration technique. Particles with surface imidazole and amine groups and particles with surface carboxylate groups have enhanced basicity and acidity, respectively. Dihydroimidazole-modified silica had greater surface basicity than the amine-modified silica. The effect on basicity and acidity increases as the amount of added functionalized silane increases. However, this increase is nonlinear with respect to the increase in added functionalized silane. Particles with both surface dihydroimidazole and carboxylate groups demonstrated reduced surface basicity and acidity.     

Environment effects on the CO vibrational shifts in erbium complexes: a quantum chemical study

The stability of lanthanide complexes and the efficiency of the energy transfer process, which makes these molecules interesting materials for technological applications, are correlated to the chemical environment surrounding the metal ion. In particular the efficiency depends on the relative position of the antenna (the ligand moiety that acts as photon absorption center) and the lanthanide ion (the emitting center), while the stability of the complex is correlated to the strength of the coordination between the rare earth and the ligands. For these reasons, knowledge of the structural properties of the complex is an interesting task to achieve. Since a large number of ligand structures hold the carboxylate group (COO(-)), which is used as an anchor for binding the antennae to the lanthanide ion, in this work we will show how the vibrational shifts of this group, induced by the interactions between the carboxylate moiety and the metal center of the lanthanide complex, can be used for obtaining in a simple way information on the structure of the chemical environment surrounding the lanthanide ion.     

Electrospray ionization ion mobility spectrometry of carboxylate anions: ion mobilities and a mass-mobility correlation

A number of carboxylate anions spanning a mass range of 87-253 amu (pyruvate, oxalate, malonate, maleate, succinate, malate, tartarate, glutarate, adipate, phthalate, citrate, gluconate, 1,2,4-benzenetricarboxylate, and 1,2,4,5-benzenetetracarboxylate) were investigated using electrospray ionization ion mobility spectrometry. Measured ion mobilities demonstrated a high correlation between mass and mobility in both N2 and CO2 drift gases. Such a strong mass-mobility correlation among structurally dissimilar ions suggests that the carboxylate functional group that these ions have in common is the source of the correlation. Computational analysis was performed to determine the most stable conformation of the studied carboxylate anions in the gas phase under the current experimental conditions. This analysis indicated that the most stable conformations for multicarboxylate anions included intramolecular hydrogen-bonded ring structures formed between the carboxylate group and the neutral carboxyl group. The carboxylate anions that form ring confirmations generally show higher ion mobility values than those that form extended conformations. This is the first observation of intramolecular hydrogen-bonded ring conformation of carboxylate anions in the gas phase at atmospheric pressure.     

Hydrolysis of Amides Catalyzed by 4‐Heterocyclohexanones: Small Molecule Mimics of Serine Proteases

The base‐promoted hydrolysis of amide substrates that contain a thiol substituent in the position α to the amide carbonyl group is effectively catalyzed by 4‐heterocyclohexanones [Eq. (1)]. The proposed mechanism of the hydrolysis reaction mimics that employed by serine proteases, and involves equilibrium binding of the substrate to the catalyst, formation of an acyl‐catalyst intermediate, and deacylation of the intermediate to release the product and regenerate the catalyst.