Single-molecule Force Spectroscopy Approach to Enzyme Catalysis

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed.     

Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate

The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys⁸²) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys⁸⁷ was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UV-visible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys⁸². These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis.     

Anion binding studies of tris(2-aminoethyl)amine based amide receptors with nitro functionalized aryl substitutions: A positional isomeric effect

Syntheses and crystal structures of tren-based amide, L¹, N,N′,N″-tris[(2-amino-ethyl)-4-nitro-benzamide] and L², N,N′,N″-tris[(2-amino-ethyl)-2-nitro-benzamide] are reported and compared with previously published tripodal amide receptor L³, N,N′,N″-tris[(2-amino-ethyl)-3-nitro-benzamide]. The crystallographic results show intramolecular and intermolecular hydrogen-bonding interactions between two arms of the tripodal receptor and two other adjacent molecules in cases of L¹ and L² whereas in addition to the above interactions an aromatic π···π stacking among tripodal arms is also observed in L³. Receptors L¹, L² and L³ having electron withdrawing -NO₂ substituted (para, ortho and meta, respectively) phenyl moieties are explored toward their solution state anion binding properties and selectivity studies. The substantial changes in chemical shifts are observed for the amide protons (-NH) and aromatic protons (-CH) with F⁻ and Cl⁻ in cases of L¹ and L³, and only with F⁻ for L², indicating the participation of -NH and -CH protons in the solution state binding events. Binding constants for the above cases are calculated by ¹H NMR titration upon monitoring the -NH signal. Receptor L² shows exclusive selectivity toward F⁻ in dimethyl sulfoxide (DMSO). The structural aspects of binding I⁻, ClO₄⁻ and SiF₆²⁻ with the monoprotonated L¹, L¹H⁺·I⁻·DMF (1), L¹H⁺·ClO₄⁻·DMF (2) and L¹H⁺·0.5SiF₆²⁻·H₂O (3), respectively are examined crystallographically. Anion binding with multiple receptor units is observed via amide N-H···anion as well as aryl C-H···anion hydrogen-bonding interactions in all the complexes as observed in cases of previously reported crystal structures of anionic complexes of protonated L³. The aryl group having nitro functionality that contributes to solution state anion binding with the neutral receptor and solid state coordination in complexes 1-3 through CH···anion interactions is noteworthy.     

Novel Thioredoxin-related Transmembrane Protein TMX4 Has Reductase Activity

In the endoplasmic reticulum (ER), a number of thioredoxin (Trx) superfamily proteins are present to enable correct disulfide bond formation of secretory and membrane proteins via Trx-like domains. Here, we identified a novel transmembrane Trx-like protein 4 (TMX4), in the ER of mammalian cells. TMX4, a type I transmembrane protein, was localized to the ER and possessed a Trx-like domain that faced the ER lumen. A maleimide alkylation assay showed that a catalytic CXXC motif in the TMX4 Trx-like domain underwent changes in its redox state depending on cellular redox conditions, and, in the normal state, most of the endogenous TMX4 existed in the oxidized form. Using a purified recombinant protein containing the Trx-like domain of TMX4 (TMX4-Trx), we confirmed that this domain had reductase activity in vitro. The redox potential of this domain (−171.5 mV; 30 °C at pH 7.0) indicated that TMX4 could work as a reductase in the environment of the ER. TMX4 had no effect on the acceleration of ER-associated degradation. Because TMX4 interacted with calnexin and ERp57 by co-immunoprecipitation assay, the role of TMX4 may be to enable protein folding in cooperation with these proteins consisting of folding complex in the ER.     

QSOX contains a pseudo-dimer of functional and degenerate sulfhydryl oxidase domains

Quiescin sulfhydryl oxidase (QSOX) catalyzes formation of disulfide bonds between cysteine residues in substrate proteins. Human QSOX1 is a multi-domain, monomeric enzyme containing a module related to the single-domain sulfhydryl oxidases of the Erv family. A partial QSOX1 crystal structure reveals a single-chain pseudo-dimer mimicking the quaternary structure of Erv enzymes. However, one pseudo-dimer “subunit” has lost its cofactor and catalytic activity. In QSOX evolution, a further concatenation to a member of the protein disulfide isomerase family resulted in an enzyme capable of both disulfide formation and efficient transfer to substrate proteins.     

Minimizing false positives in kinase virtual screens

In spite of recent improvements in docking and scoring methods, high false-positive rates remain a common issue in structure-based virtual screening. In this study, the distinctive features of false positives in kinase virtual screens were investigated. A series of retrospective virtual screens on kinase targets was performed on specifically designed test sets, each combining true ligands and experimentally confirmed inactive compounds. A systematic analysis of the docking poses generated for the top-ranking compounds highlighted key aspects differentiating true hits from false positives. The most recurring feature in the poses of false positives was the absence of certain key interactions known to be required for kinase binding. A systematic analysis of 444 crystal structures of ligand-bound kinases showed that at least two hydrogen bonds between the ligand and the backbone protein atoms in the kinase hinge region are present in 90% of the complexes, with very little variability across targets. Closer inspection showed that when the two hydrogen bonds are present, one of three preferred hinge-binding motifs is involved in 96.5% of the cases. Less than 10% of the false positives satisfied these two criteria in the minimized docking poses generated by our standard protocol. Ligand conformational artifacts were also shown to contribute to the occurrence of false positives in a number of cases. Application of this knowledge in the form of docking constraints and post-processing filters provided consistent improvements in virtual screening performance on all systems. The false-positive rates were significantly reduced and the enrichment factors increased by an average of twofold. On the basis of these results, a generalized two-step protocol for virtual screening on kinase targets is suggested.     

Analysis of disulphide bond connectivity patterns in protein tertiary structure

The analysis of disulphide bond containing proteins in the Protein Data Bank (PDB) revealed that out of 27,209 protein structures analyzed, 12,832 proteins contain at least one intra-chain disulphide bond and 811 proteins contain at least one inter-chain disulphide bond. The intra-chain disulphide bond containing proteins can be grouped into 256 categories based on the number of disulphide bonds and the disulphide bond connectivity patterns (DBCPs) that were generated according to the position of half-cystine residues along the protein chain. The PDB entries corresponding to these 256 categories represent 509 unique SCOP superfamilies. A simple web-based computational tool is made freely available at the website http://www.ccmb.res.in/bioinfo/dsbcp that allows flexible queries to be made on the database in order to retrieve useful information on the disulphide bond containing proteins in the PDB. The database is useful to identify the different SCOP superfamilies associated with a particular disulphide bond connectivity pattern or vice versa. It is possible to define a query based either on a single field or a combination of the following fields, i.e., PDB code, protein name, SCOP superfamily name, number of disulphide bonds, disulphide bond connectivity pattern and the number of amino acid residues in a protein chain and retrieve information that match the criterion. Thereby, the database may be useful to select suitable protein structural templates in order to model the more distantly related protein homologs/analogs using the comparative modeling methods.     

Multinuclear magnetic resonance, electrospray ionization-mass spectroscopy, and parametric method 5 studies of a new derivative of gossypol with 2-thiophenecarbohydrazide as well as its complexes with LI+, Na+, K+, RB+, and Cs+ cations

A new derivative of racemic gossypol with 2-thiophenecarbohydrazide (GHHT) and its complexes with monovalent cations have been synthesized and studied by electrospray ionization-mass spectroscopy (ESI-MS), multinuclear nuclear magnetic resonance (NMR), as well as by the Parametric Method 5 (PM5) methods. It is demonstrated that GHHT forms stable complexes of 1:1 stoichiometry with monovalent metal cations. The structures of the complexes are stabilized by three types of intramolecular hydrogen bonds. The spectroscopic methods have provided clear evidence that GHHT and its complexes exist in the DMSO-d6 solution in the N-imine-N-imine tautomeric forms. The structures of the GHHT and its complexes with Li+, Na+, K+, Rb+, and Cs+ cations are visualized and discussed in detail.     

Monensin A methyl ester complexes with Li+, Na+, and K+ cations studied by ESI-MS, 1H- and 13C-NMR, FTIR, as well as PM5 semiempirical method

Monensin A methyl ester (MON1) was synthesized by a new method and its ability to form complexes with Li+, Na+, and K+ cations was studied by electrospray ionization-mass spectroscopy (ESI-MS), 1H and 13C nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), and PM5 semiempirical methods. It is shown that MON1 with monovalent metal cations forms stable complexes of 1:1 stoichiometry. The structures of the complexes are stabilized by intramolecular hydrogen bonds in which the OH groups are always involved. In the structure of MON1, the oxygen atom of the C=O ester group is involved in very weak bifurcated intramolecular hydrogen bonds with two hydroxyl groups, whereas in the complexes of MON1 with monovalent metal cations the C=O ester group is not engaged in any intramolecular hydrogen bonds. Furthermore, it is demonstrated that the strongest intramolecular hydrogen bonds are formed within the MON1-Li+ complex structure. The structures of the MON1 and its complexes with Li+, Na+, and K+ cations are visualized and discussed in detail.