Models For Enzyme Active Sites Research Articles

Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases.

Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy.

A DFT study on the catalytic mechanism of UDP-glucose dehydrogenase

Uridine 5′-diphosphate glucuronic acid (UDPGlcUA) is a key intermediary metabolite in many species, including pathogenic bacteria and humans. It is biosynthesized from UDP-glucose (UDPGlc) by uridine diphosphate glucose dehydrogenase (UDPGlcDH) via a twofold two-electron–one-proton oxidation that successively transforms the 6-hydroxymethyl of glucopyranose into a formyl, and the latter into the final carboxylic function. The catalytic mechanism of UDPGlcDH was investigated using a large enzyme active-site model in combination with the B3LYP method and the polarizable continuum model (IEF-PCM) self-consistent reaction field. The latter was used to correct for the long-range electrostatic effect of the protein environment. The overall mechanism consists of four catalytic steps: (i) NAD+-dependent oxidation of glucose to glucuronaldehyde, (ii) nucleophilic addition of Cys260–SH to glucuronaldehyde to form a 6-thiohemiacetal intermediate, (iii) NAD+-dependent oxidation of the 6-thiohemiacetal to form a 6-thioester intermediate, and finally, (iv) hydrolysis of the 6-thioester to give glucuronic acid. In addition, this study also provides insight into the debated roles of Lys204 and Asp264, and the most likely protonation state of a reactive Michaelis complex of UDPGlcDH.

Synthesis, structural studies and solubility properties of zinc(II), nickel(II) and copper(II) complexes of bulky tris(triazolyl)borate ligands

Tris(triazolyl)borate (Ttz) ligands are sterically similar to tris(pyrazolyl)borate (Tp) but complexes of Ttz show improved solubility in water and alcohols due to their propensity for forming hydrogen bonds. Recently developed bulky tris(triazolyl)borate ligands can produce four and five coordinate transition metal complexes and serve as models for enzyme active sites in an aqueous environment. Herein we report the synthesis of such complexes, i.e. (Ttz t Bu,Me)ZnCl, (Ttz t Bu,Me)ZnBr, (Ttz t Bu,Me)NiCl, and (Ttz t Bu,Me)CuCl, which were analyzed by X-ray crystallographic and spectroscopic methods [Ttz t Bu,Me = tris(3- t-butyl-5-methyl-1,2,4-triazolyl)borate]. (Ttz t Bu,Me)ZnCl crystallizes as two different polymorphs with cubic and monoclinic symmetry. Both polymorphs of (Ttz t Bu,Me)ZnCl and (Ttz t Bu,Me)ZnBr have tetrahedral zinc atoms whereas the geometries at the metal in (Ttz t Bu,Me)NiCl and (Ttz t Bu,Me)CuCl are distorted tetrahedral. All complexes are methanol soluble and they also dissolve in methanol/water mixtures with up to 60% water.

Thermodynamics of binding of angiotensin-converting enzyme inhibitors to enzyme active site model

The density functional theory (DFT) using Becke3LYP functional and the two-layered ONIOM Becke3LYP:MNDO calculations have been carried out to investigate the structural and thermodynamic properties of 29 neutral and deprotonated angiotensin-converting enzyme inhibitors (H 2O, enalaprilat, cilazaprilat, imidaprilat, perindoprilat, quinaprilat, ramiprilat, spiraprilat, trandolaprilat, fosinoprilat, omapatrilat, captopril, zofenoprilat, silanediol and keto-ACE) in complex with zinc cation and three first-shell ligands as models of active site of angiotensin-converting enzyme. The influence of deprotonation on the structure and relative energetics of model complexes was examined. Interaction enthalpies and Gibbs energies between inhibitors and angiotensin-converting enzyme active site model were calculated. The structure and thermodynamics of optimized complexes are discussed from the point of view of their biological importance.

Design and Synthesis of Polymetallic Complexes Based on meso-Calix[4]pyrrole: Platforms for Multielectron Chemistry

A first series of highly metalated calix[4]pyrrole derivatives has been obtained by treating calix[4]pyrrole with [RuCp*(CH3CN)3]PF6 in THF under reflux for 4 h to give the corresponding tetrakisruthenocene-like product and then with Cu(OTf)2 in the presence of triethylamine to give the corresponding copper(II) complex. This latter product contains five metal centers supported by a core ligand consisting of 32 non-hydrogen atoms. Electrochemical and ESR spectroscopic analyses provide support for the notion that both complexes are capable of transferring four electrons reversibly at a potential of 0.95 V vs SCE. Related studies involving the copper complex reveal the presence of an additional reversible reduction at −0.34 V vs SCE that is ascribed to a Cu(II)/Cu(I) couple. These findings make these systems of potential interest as simple-to-prepare electron reservoirs and as possible models for enzyme active sites.

Sterically Bulky Tris(triazolyl)borate Ligands as Water-Soluble Analogues of Tris(pyrazolyl)borate

The recently synthesized 3-tert-butyl-5-methyl-1,2,4-triazole reacted with KBH4 to give the new potassium tris(3-tert-butyl-5-methyl-1,2,4-triazolyl)borate K(Ttz(tBu,Me)) ligand. Ttz(tBu,Me) formed a four-coordinate (Ttz(tBu,Me))CoCl complex and five-coordinate (Ttz(tBu,Me))CoNO3 and (Ttz(tBu,Me))ZnOAc complexes. When these complexes were compared to their Tp(tBu,Me) analogues, it was found that Ttz(tBu,Me) resulted in negligible steric differences. K(Ttz(tBu,Me)) is more water-soluble than K(Tp(tBu,Me)), so bulky tris(triazolyl)borate ligands should lead to functional models for enzyme active sites in an aqueous environment and the creation of water-soluble analogues of Tp catalysts.

Proton transfer dynamics and N?H bond lengthening in N?H���N model systems: a solid-state NMR study

Hydrogen-bonded pairs of the 2-methylimidazolium cation and 2-methylimidazole are expected to have matched pKa values, owing to chemical symmetry. These structures serve as models for enzyme active sites. Several crystalline salts of this system were prepared and characterized by crystallography and solid-state NMR. Short N···N distances are observed (∼2.65 Å), and fast (>105 s−1) proton transfer through the N—H···N bridge was suggested by NMR lineshape measurements at temperatures from 200 to 320 K. The equilibrium constants for these transfer processes were found to differ from unity, and to be strongly temperature and counter-ion dependent. For the perchlorate salt, the proton is observed to be mainly attached to one of the bridge nitrogens across this temperature range, for the iodide salt, the proton is substantially delocalized on to both bridging nitrogens across the temperature range. Interestingly, for the chloride and bromide salts, a temperature-dependent equilibrium constant is observed, with equal populations of the two isomers in rapid exchange at room temperature and an effectively trapped proton (or very strong population of one isomer) at 200 K. This temperature-dependent equilibrium constant indicates that the proton transfer is associated with an enthalpy of the order the 20 kJ mol−1. This study underscores the power of NMR spectroscopy to account for protons, and the important influence of remote ionic interactions on proton transfer coordinates. Copyright © 2001 John Wiley & Sons, Ltd.

Linking CoMFA and protein homology models of enzyme–inhibitor interactions: an application to non-steroidal aromatase inhibitors

An approach to compare quantitatively a ligand-based (CoMFA) model and an enzyme active site model was investigated. The active site of the cytochrome P450 human aromatase was constructed by homology modeling techniques and two structurally different non-steroidal aromatase inhibitors were docked into it. A CoMFA model was then developed on a related series of non-steroidal inhibitors by correlating their inhibitory activity (expressed as −log IC 50 values) versus only 11 steric descriptors (i.e. C sp3–ligand steric interaction energies). The resulting 3D-QSAR coefficients (11) and the steric field values of the aromatase active site calculated at the same points of the CoMFA lattice (i.e. eleven C sp3–protein steric interaction energies) were pair-wise compared. Specifically, when a positive coefficient was associated with a negative or low (<5 kcal/mol) value of the protein steric field or, alternatively, a negative coefficient was associated with a large positive value of the protein steric field we recorded as many matches. When a 3D-QSAR coefficient did not correspond to the protein steric potential in the sense described above we considered that point as a mis-matching point. In our view, in spite of several limitations, such a comparison represents a valuable criterion to evaluate quantitatively how convergent are the results from a 3D-QSAR CoMFA model and a homology-built protein 3D structure.

Active site model of cyclohexanone monooxygenase. 3. The case of benzyl sulfides bearing polar groups

The stereopreference of cyclohexanone monooxygenase for a series of benzyl methyl sulfides bearing in the p-position groups with different σ p values has been rationalized by locating inside the enzyme active site model electron-rich and electron-poor regions.

Enzymic hydrolysis of ethyl 3-hydroxy-3-phenylpropanoate: observations on an enzyme active-site model

Enzymic hydrolysis of ethyl 3-hydroxy-3-phenylpropanoate: observations on an enzyme active-site model

Subsite-specific reactivity of Fe 4S 4 clusters bound to tridentate, trithiol ligands as models for enzyme active sites

Subsite-specific reactivity of Fe 4S 4 clusters bound to tridentate, trithiol ligands as models for enzyme active sites

Oxygen atom transfer reactions of molybdenum complexes and iron-porphyrin complexes as models for enzyme active sites

Oxygen atom transfer reactions of molybdenum complexes and iron-porphyrin complexes as models for enzyme active sites