Mapping enzyme active sites in complex proteomes

Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3 beta-hydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites.     

Quantum mechanical design of enzyme active sites

The design of active sites has been carried out using quantum mechanical calculations to predict the rate-determining transition state of a desired reaction in presence of the optimal arrangement of catalytic functional groups (theozyme). Eleven versatile reaction targets were chosen, including hydrolysis, dehydration, isomerization, aldol, and Diels-Alder reactions. For each of the targets, the predicted mechanism and the rate-determining transition state (TS) of the uncatalyzed reaction in water is presented. For the rate-determining TS, a catalytic site was designed using naturalistic catalytic units followed by an estimation of the rate acceleration provided by a reoptimization of the catalytic site. Finally, the geometries of the sites were compared to the X-ray structures of related natural enzymes. Recent advances in computational algorithms and power, coupled with successes in computational protein design, have provided a powerful context for undertaking such an endeavor. We propose that theozymes are excellent candidates to serve as the active site models for design processes.     

Quantum chemical modeling of enzyme active sites and reaction mechanisms

Density functional methods, in particular the B3LYP functional, together with the explosive enhancement of computational power, have in the last 5 years or so made it possible to model enzyme active sites and reaction mechanisms in a quite realistic way. Many mechanistic problems have indeed been addressed and solved. This review gives a brief account of the methods and models used to study enzyme active sites and their reaction mechanisms using quantum chemical methods. Examples are given from our recent work in this field. Future perspectives of the field are discussed.     

Novel cofactors via post-translational modifications of enzyme active sites

Recent crystallographic and biochemical studies have revealed the existence of numerous novel post-translational modifications within enzyme active sites. These modifications create structural and functional diversity. Although the function and biosynthesis of some of these modifications are well understood, others need further investigation.     

Comparison of substructural epitopes in enzyme active sites using self-organizing maps

Summary This paper presents a new algorithm to compare substructural epitopes in protein binding cavities. Through the comparison of binding cavities accommodating well characterized ligands with cavities whose actual guests are yet unknown, it is possible to draw some conclusions on the required shape of a putative ligand likely to bind to the latter cavities. To detect functional relationships among proteins, their binding-site exposed physicochemical characteristics are described by assigning generic pseudocenters to the functional groups of the amino acids flanking the particular active site. The cavities are divided into small local regions of four pseudocenters having the shape of a pyramid with triangular basis. To find similar local regions, an emergent self-organizing map is used for clustering. Two local regions within the same cluster are similar and form the basis for the superpositioning of the corresponding cavities to score this match. First results show that the similarities between enzymes with the same EC number can be found correctly. Enzymes with different EC numbers are detected to have no common substructures. These results indicate the benefit of this method and motivate further studies.     

Factors determining the enzyme catalytic power caused by noncovalent interactions: Charge alterations in enzyme active sites

Understanding the origin of the enormous catalytic power of enzymes is very important. Electrostatic interactions and desolvation are the phenomena that are most proposed to explain the catalysis of enzymes; however, they also decelerate enzymatic reactions. How enzymes catalyze reactions through noncovalent interactions is still not well-understood. In this study, we explored how enzyme-substrate noncovalent interactions affect the free energy barriers (ΔG³s) of reactions by using a theoretical derivation approach. We found that enzymes reduce ΔG³s of reactions by decreasing positive charges and/or increasing negative charges in the electron-donating centers and by decreasing negative charges and/or increasing positive charges in the electron-accepting centers of reactions. Enzyme-substrate noncovalent interactions are essential approaches through which the charge alterations lead to ΔG³ reductions. Validations with reported experimental data demonstrated that this charge alteration mechanism can explain the catalyses caused by diverse types of noncovalent interactions. Electrostatic interactions and desolvation are the most observed noncovalent interactions essential for ΔG³ reductions. This mechanism does not contradict any specific enzymatic catalysis and overcomes the shortages of the electrostatic interaction and desolvation mechanisms. This study can provide useful guidance in exploring enzymatic catalysis and designing catalyst.     

Effect of solvation water shells on enzyme active sites in zinc-dependent hydrolases

Structural Chemistry - In this work, we focus on some structural aspects of enzyme catalysis by modeling effects of the electrostatic potential created by the solvation water shells on properties...     

Triazacyclophane (TAC)-scaffolded histidine and aspartic acid residues as mimics of non-heme metalloenzyme active sites

We describe the synthesis and coordination behaviour to copper(II) of two close structural triazacyclophane-based mimics of two often encountered aspartic acid and histidine containing metalloenzyme active sites. Coordination of these mimics to copper(I) and their reaction with molecular oxygen leads to the formation of dimeric bis(μ-hydroxo) dicopper(II) complexes.     

Algorithms of the flexible effective fragment method used for modeling of transformations in enzyme active sites

In the present work, the algorithms and software implementations of a combined quantum and molecular mechanics method in terms of conformationally flexible effective fragments are discussed. This method is widely used for computations of energy profiles in enzyme catalysis.     

Structure-based drug design: exploring the proper filling of apolar pockets at enzyme active sites

The proper filling of apolar pockets at enzyme active sites is central for increasing binding activity and selectivity of hits and leads in medicinal chemistry. In our structure-based design approach toward the generation of potent enzyme inhibitors, we encountered a variety of challenges in gaining suitable binding affinity from the occupation of such pockets. We summarize them here for the first time. A fluorine scan of tricyclic thrombin inhibitors led to the discovery of favorable orthogonal dipolar C-F...CO interactions. Efficient cation-pi interactions were established in the S4 pocket of factor Xa, another serine protease from the blood coagulation cascade. Changing from mono- to bisubstrate inhibitors of catechol O-methyltransferase, a target in the L-Dopa-based treatment of Parkinson's disease, enabled the full exploitation of a previously unexplored hydrophobic pocket. Conformational preorganization of a pocket at an enzyme active site is crucial for harvesting binding affinity. This is demonstrated for two enzymes from the nonmevalonate pathway of isoprenoid biosynthesis, IspE and IspF, which are pursued as antimalarial targets. Disrupting crystallographically defined water networks on the way into a pocket might cost all of the binding free enthalpy gained from its occupation, as revealed in studies with tRNA-guanine transglycosylase, a target against shigellosis. Investigations of the active site of plasmepsin II, another antimalarial target, showed that principles for proper apolar cavity filling, originally developed for synthetic host-guest systems, are also applicable to enzyme environments.     

Structural reorganization and preorganization in enzyme active sites: comparisons of experimental and theoretically ideal active site geometries in the multistep serine esterase reaction cycle

Many enzymes catalyze reactions with multiple chemical steps, requiring the stabilization of multiple transition states during catalysis. Such enzymes must strike a balance between the conformational reorganization required to stabilize multiple transition states of a reaction and the confines of a preorganized active site in the polypeptide tertiary structure. Here we investigate the compromise between structural reorganization during the catalytic process and preorganization of the active site for a multistep enzyme-catalyzed reaction, the hydrolysis of esters by the Ser-His-Asp/Glu catalytic triad. Quantum mechanical transition states were used to generate ensembles of geometries that can catalyze each individual step in the mechanism. These geometries are compared to each other by superpositions of catalytic atoms to find "consensus" geometries that can catalyze all steps with minimal rearrangement. These consensus geometries are found to be excellent matches for the natural active site. Preorganization is therefore found to be the major defining characteristic of the active site, and reorganizational motions often proposed to promote catalysis have been minimized. The variability of enzyme active sites observed by X-ray crystallography was also investigated empirically. A catalog of geometrical parameters relating active site residues to each other and to bound inhibitors was collected from a set of crystal structures. The crystal-structure-derived values were then compared to the ranges found in quantum mechanically optimized structures along the entire reaction coordinate. The empirical ranges are found to encompass the theoretical ranges when thermal fluctuations are taken into account. Therefore, the active sites are preorganized to a geometry that can be objectively and quantitatively defined as minimizing conformational reorganization while maintaining optimal transition state stabilization for every step during catalysis. The results provide a useful guiding principle for de novo design of enzymes with multistep mechanisms.     

Effects of acid etching on the structure of PtNi catalyst and total exposed active sites

The integration technology of hydrogen preparation–hydrogen storage not only can utilize hydrogen energy efficiently but also can improve the selectivity of the electrode maximally. In the present work, the structure and composition of the PtNi catalyst was characterized by X-ray diffraction (XRD); and its electrochemical properties, morphology, and surface binding energy were analyzed by cyclic voltammetry (CV) and linear scanning voltammetry (LSV), scanning electron microscopy equipped with energy-dispersive spectrometry (SEM-EDS), and X-ray photoelectron spectroscopy (XPS), respectively. The effects of different acid etching treatments (e.g., etching time, etchant concentration, and etching temperature) on the structure and surface active sites were investigated by the orthogonal experiment. The experimental results reveal that after etching with 0.5 mol/L of perchloric acid for 0.5 h at 60°C, the electrode weight loss of the PtNi catalyst is mainly attributed to the large loss of Ni atoms in film layer. This results in the reduced alloy phase in film layer and the appearance of Pt characteristic diffraction peak. The relative content of Pt on the surface of the film electrode increases significantly, and the total number of active sites also increases correspondingly. The binding energy of Pt4f_7/2 decreases by 0.19 eV, and the number of active sites involved in hydrogen release decreases, indicative of the reduced promotion effect of the PtNi catalyst on hydrogen release.     

Design and synthesis of a novel triptycene-based ligand for modeling carboxylate-bridged diiron enzyme active sites

A novel triptycene-based ligand with a preorganized framework was designed to model carboxylate-bridged diiron active sites in bacterial multicomponent monooxygenase (BMM) hydroxylase enzymes. The synthesis of the bis(benzoxazole)-appended ligand L1 depicted was accomplished in 11 steps. Reaction of L1 with iron(II) triflate and a carboxylate source afforded the desired diiron(II) complex [Fe(2)L1(μ-OH)(μ-O(2)CAr(Tol))(OTf)(2)].     

On the structure of surface active centers on CuO

It has been established that surface active centers on CuO can be associated with its surface defects, i.e. surface exits of twinning and screw dislocations where highly reactive oxygen and CO can be coordinated.

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Hydrogen-bonding interactions in the active sites of cytochrome P450cam and its site-directed mutants.

Resonance Raman spectroscopy is applied to the cyanide adducts of cytochrome P450cam and its T252A and D251N site-directed mutants, both in their substrate-free and camphor-bound forms, to probe active-site heme structure and, in particular, interactions of the FeCN fragment with potential active-site H-bond donors. In contrast to the ferrous CO and ferric NO adducts, which form only essentially linear (slightly distorted) FeXY fragments, the spectra of the ferric CN(-) adducts provide clear evidence the for the existence of an additional, rather highly bent, conformer; that is, the cyanide complexes form both linear and bent conformers in both the substrate-free and substrate-bound forms. Formation of this bent conformer is most reasonably attributed to the presence of off-axis H-bond donors, which induce distortion on the FeCN fragment but not the FeCO and FeNO fragments, which are poorer H-bond acceptors. For all three proteins, the substrate-free form exhibits a complex spectral pattern which arises because one of the modes associated with the FeCN fragment is coupled with two heme macrocycle deformation modes. Significantly, no evidence for such coupling is observed in the spectra of the camphor-bound forms. While various unknown factors may possibly give rise to selective activation of such coupling in the substrate-free derivative, given the known facts about the active-site architecture of this enzyme, a plausible explanation is that the bent conformer is oriented toward the water-filled substrate-binding site in the substrate-free form, but oppositely, toward the proposed proton delivery shuttle, in the substrate-bound form. Sensitivity of the FeCN modes to H(2)O/D(2)O exchange in the two camphor-bound mutants, which is apparently absent for the camphor-bound native protein, is most reasonably attributed to the known presence of extra water in the active sites of these mutants.     

Rapid screening by MALDI-TOF mass spectrometry to probe binding specificity at enzyme active sites

The binding affinity of aspartate decarboxylase has been probed using MALDI-TOF spectrometry; adducts formed covalently in the active site were detected by MALDI-TOF mass spectrometry after incubation of the enzyme with a range of potential ligands in the presence of NaCNBH3; this has highighted key structural features which will aid design of potential inhibitors.     

The solution structure of [Cu(aq)]2+ and its implications for rack-induced bonding in blue copper protein active sites

The structure of [Cu(aq)]2+ has been investigated by using full multiple-scattering theoretical (MXAN) analysis of the copper K-edge X-ray absorption (XAS) spectrum and density functional theory (DFT) to test both ideal Td and square-planar four-coordinate, five-coordinate square-pyramidal, and six-coordinate octahedral [Cu(aq)]2+ models. The best fit was an elongated five-coordinate square pyramid with four Cu-O(eq) bonds (2 x 1.98 +/- 0.03 A and 2 x 1.95 +/- 0.03 A) and a long Cu-O(ax) bond (2.35 +/- 0.05 A). The four equatorial ligands were D2d-distorted from the mean equatorial plane by +/-(17 +/- 4) degrees, so that the overall symmetry of [Cu(H2O)5]2+ is C2v. The four-coordinate MXAN fit was nearly as good, but the water ligands (4 x 1.96 +/- 0.02 A) migrated +/-(13 +/- 4) degrees from the mean equatorial plane, making the [Cu(H2O)4]2+ model again D2d-distorted. Spectroscopically calibrated DFT calculations were carried out on the C2v elongate square-pyramidal and D2d-distorted four-coordinate MXAN copper models, providing comparative electronic structures of the experimentally observed geometries. These calculations showed 0.85e spin on Cu(II) and 0.03e electron spin on each of the four equatorial water oxygens. All covalent bonding was restricted to the equatorial plane. In the square-pyramidal model, the electrostatic Cu-O(ax) bond was worth only 96.8 kJ mol(-1), compared to 304.6 kJ mol(-1) for each Cu-O(eq) bond. Both MXAN and DFT showed the potential well of the axial bond to be broad and flat, allowing large low-energy excursions. The irregular geometry and D2d-distorted equatorial ligand set sustained by unconstrained [Cu(H2O)5]2+ warrants caution in drawing conclusions regarding structural preferences from small molecule crystal structures and raises questions about the site-structural basis of the rack-induced bonding hypothesis of blue copper proteins. Further, previously neglected protein folding thermodynamic consequences of the rack-bonding hypothesis indicate an experimental disconfirmation.