High-energy Intermediates Research Articles

Predicting Substrates by Docking High-Energy Intermediates to Enzyme Structures

With the emergence of sequences and even structures for proteins of unknown function, structure-based prediction of enzyme activity has become a pragmatic as well as an interesting question. Here we investigate a method to predict substrates for enzymes of known structure by docking high-energy intermediate forms of the potential substrates. A database of such high-energy transition-state analogues was created from the KEGG metabolites. To reduce the number of possible reactions to consider, we restricted ourselves to enzymes of the amidohydrolase superfamily. We docked each metabolite into seven different amidohydrolases in both the ground-state and the high-energy intermediate forms. Docking the high-energy intermediates improved the discrimination between decoys and substrates significantly over the corresponding standard ground-state database, both by enrichment of the true substrates and by geometric fidelity. To test this method prospectively, we attempted to predict the enantioselectivity of a set of chiral substrates for phosphotriesterase, for both wild-type and mutant forms of this enzyme. The stereoselectivity ratios of the six enzymes considered for those four substrate enantiomer pairs differed over a range of 10- to 10,000-fold and underwent 20 switches in stereoselectivities for favored enantiomers, compared to the wild type. The docking of the high-energy intermediates correctly predicted the stereoselectivities for 18 of the 20 substrate/enzyme combinations when compared to subsequent experimental synthesis and testing. The possible applications of this approach to other enzymes are considered.

Use of iridium oxide films as hydrogen gas evolution cathodes in aqueous media

Hydrous iridium oxide films are highly resistant to reduction under cathodic, hydrogen gas evolution, conditions in aqueous acid or base. Such behavior is not in agreement with simple thermodynamic (Pourbaix) data based on the assumption that the system behaves in a reversible manner. The barrier to reduction is attributed, as discussed earlier for RuO2, to the involvement of high-energy intermediates (iridium atoms or microclusters of same) which can only be generated at unusually negative overpotential values evidently far into the hydrogen gas evolution region. Thermally prepared IrO2/Ti electrodes are possible candidates for hydrogen gas evolution cathodes in water electrolysis cells; however, under extended operating conditions, the performance of these cathodes was found to deteriorate due to gradual shedding of the active oxide layer.

Synthesis of Methylene- and Difluoromethylenephosphonate Analogues of Uridine-4-phosphate and 3-Deazauridine-4-phosphate

Cytidine triphosphate synthetase (CTPS) catalyzes the formation of cytidine triphosphate from glutamine, uridine-5'-triphosphate (UTP), and adenosine-5'-triphosphate. Inhibitors of CTPS are of interest because of their potential as therapeutic agents. One approach to potent enzyme inhibitors is to use analogues of high energy intermediates formed during the reaction. The CTPS reaction proceeds via the high energy intermediate UTP-4-phosphate (UTP-4-P). Four novel analogues of uridine-4-phosphate (U-4-P) and 3-deazauridine-4-phosphate (3-deazaU-4-P) were synthesized in which the labile phosphate ester oxygen was replaced with a methylene and difluoromethylene group. The methylene analogue of U-4-P, compound 1, was prepared by a reaction of the sodium salt of tert-butyl diethylphosphonoacetate with protected, 4-O-activated uridine followed by acetate deprotection and decarboxylation. It was found that this compound undergoes relatively facile dephosphonylation presumably via a metaphosphate intermediate. The difluoromethylene derivative, compound 2, was prepared by electrophilic fluorination of protected 1. This compound was stable and did not undergo dephosphonylation. Synthesis of the methylene analogue of 3-deazaU-4-P, compound 3, was achieved by ribosylation of protected 4-(phosphonomethyl)-2-hydroxypyridine. Electrophilic fluorination was also employed in the preparation of protected 4-(phosphonodifluoromethyl)-2-hydroxypyridine which was used as the key building block in the synthesis of difluoro derivative 4. These compounds represent the first examples of a nucleoside in which the base has been chemically modified with a methylene or difluormethylenephosphonate group.

A Mass Spectrometry-Based Probe of Equilibrium Intermediates in Protein-Folding Reactions

Described here is a mass spectrometry- and H/D exchange-based approach for the detection of equilibrium intermediate state(s) in protein-folding reactions. The approach utilizes the stability of unpurified proteins from rates of H/D exchange (SUPREX) technique to measure the m value (i.e., delta DeltaG/delta [denaturant] value) associated with the folding reaction of a protein. Such SUPREX m-value analyses can be made over a wide range of denaturant concentrations. Thus, the described approach is well-suited for the detection of high-energy intermediates that might be populated at low denaturant concentrations and hard to detect in conventional chemical denaturation experiments using spectroscopic probes. The approach is demonstrated on four known non-two-state folding proteins, including alpha-lactalbumin, cytochrome c, intestinal fatty acid binding protein (IFABP), and myoglobin. The non-two-state folding behavior of each model protein system was detected by the described method. The cytochrome c, myoglobin, and IFABP systems each had high-energy intermediate states that were undetected in conventional optical spectroscopy-based studies and previously required other more specialized biophysical approaches (e.g., nuclear magnetic resonance spectroscopy-based methods and protease protection assays) for their detection. The SUPREX-based approach outlined here offers an attractive alternative to these other approaches, because it has the advantage of speed and the ability to analyze both purified and unpurified protein samples in either concentrated or dilute solution.

Synthesis of β-ketophosphonate analogs of glutamyl and glutaminyl adenylate, and selective inhibition of the corresponding bacterial aminoacyl-tRNA synthetases

The aminoacyl-β-ketophosphonate-adenosines (aa-KPA) are stable analogs of the aminoacyl adenylates, which are high-energy intermediates in the formation of aminoacyl-tRNA catalyzed by aminoacyl-tRNA synthetases (aaRS). We have synthesized glutamyl-β-ketophosphonate-adenosine (Glu-KPA) and glutaminyl-β-ketophosphonate-adenosine (Gln-KPA), and have tested them as inhibitors of their cognate aaRS, and of a non-cognate aaRS. Glu-KPA is a competitive inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS) with a K i of 18 μM with respect to its substrate glutamate, and binds at one site on this monomeric enzyme; the non-cognate Gln-KPA also binds this GluRS at one site, but is a much weaker ( K i = 2.9 mM) competitive inhibitor. By contrast, Gln-KPA inhibits E. coli glutaminyl-tRNA synthetase (GlnRS) by binding competitively but weakly at two distinct sites on this enzyme (average K i of 0.65 mM); the non-cognate Glu-KPA shows one-site weak ( K i = 2.8 mM) competitive inhibition of GlnRS. These kinetic results indicate that the glutamine and the AMP modules of Gln-KPA, connected by the β-ketophosphonate linker, cannot bind GlnRS simultaneously, and that one Gln-KPA molecule binds the AMP-binding site of GlnRS through its AMP module, whereas another Gln-KPA molecule binds the glutamine-binding site through its glutamine module. This model suggests that similar structural constraints could affect the binding of Glu-KPA to the active site of mammalian cytoplasmic GluRSs, which are evolutionarily much closer to bacterial GlnRS than to bacterial GluRS. This possibility was confirmed by the fact that Glu-KPA inhibits bovine liver GluRS 145-fold less efficiently than E. coli GluRS by competitive weak binding at two distinct sites (average K i = 2.6 mM). Moreover, these kinetic differences reveal that the active sites of bacterial GluRSs and mammalian cytoplasmic GluRSs have substantial structural differences that could be further exploited for the design of better inhibitors specific for bacterial GluRSs, promising targets for antimicrobial therapy.

From Supramolecular Photochemistry to Self-Assembled Photoactive Architectures: The Emergence of Photochemical Nanosciences

The use of supramolecular interactions to control and direct excited-state reactivity is described, with emphasis on the author's contributions in this area. The systems are based on the multiple-point hydrogen-bonding molecular recognition motif present in the melamine–barbituric acid system, and are designed to operate in solution phase. Despite the weak nature of non-covalent interactions, control of high energy intermediates such as excited cinnamantes, stilbenes, or fullerenes is possible. These results demonstrate the particular sensitivity of short-lived electronically excited molecules to their environment, and particularly to the mutual orientation of the reactants.

Unveiling a Hidden Folding Intermediate in c-Type Cytochromes by Protein Engineering

Several investigators have highlighted a correlation between the basic features of the folding process of a protein and its topology, which dictates the folding pathway. Within this conceptual framework we proposed that different members of the cytochrome c (cyt c) family share the same folding mechanism, involving a consensus partially structured state. Pseudomonas aeruginosa cyt c(551) (Pa cyt c(551)) folds via an apparent two-state mechanism through a high energy intermediate. Here we present kinetic evidence demonstrating that it is possible to switch its folding mechanism from two to three state, stabilizing the high energy intermediate by rational mutagenesis. Characterization of the folding kinetics of one single-site mutant of the Pa cyt c(551) (Phe(7) to Ala) indeed reveals an additional refolding phase and a fast unfolding process which are explained by the accumulation of a partially folded species. Further kinetic analysis highlights the presence of two parallel processes both leading to the native state, suggesting that the above mentioned species is a non obligatory on-pathway intermediate. Determination of the crystallographic structure of F7A shows the presence of an extended internal cavity, which hosts three "bound" water molecules and a H-bond in the N-terminal helix, which is shorter than in the wild type protein. These two features allow us to propose a detailed structural interpretation for the stabilization of the native and especially the intermediate states induced by a single crucial mutation. These results show how protein engineering, x-ray crystallography and state-of-the-art kinetics concur to unveil a folding intermediate and the structural determinants of its stability.

Membrane biophysics

Membrane biophysics

New Insights into the Drug Binding, Transport and Lipid Flippase Activities of the P-Glycoprotein Multidrug Transporter

The MDR1 P-glycoprotein, an ATP-binding cassette (ABC) superfamily member that functions as an ATP-driven drug efflux pump, has been linked to resistance of human tumors to multiple chemotherapeutic agents. P-glycoprotein binds and actively transports a large variety of hydrophobic drugs and peptides. P-glycoprotein in reconstituted proteoliposomes is also an outwardly directed flippase for membrane phospholipids and simple glycosphinglipids. This review focuses on recent advances in our understanding of P-glycoprotein structure and function, particularly through the use of fluorescence spectroscopic approaches. Progress is being made towards understanding the structure of the transporter, especially the spatial relationship between the two nucleotide-binding domains. Exploration of the P-glycoprotein catalytic cycle using vanadate-trapped complexes has revealed that drug transport likely takes place by concerted conformational changes linked to relaxation of a high energy intermediate. Low resolution mapping of the protein using fluorescence resonance energy transfer showed that both the H and R drug-binding sites are located within the cytoplasmic leaflet. Two drugs can bind to the R-site simultaneously, suggesting that the protein contains a large flexible binding region.

The Reactant State for Substrate-Activated Turnover of Acetylthiocholine by Butyrylcholinesterase is a Tetrahedral Intermediate

Secondary beta-deuterium kinetic isotope effects have been measured as a function of substrate concentration for recombinant human butyrylcholinesterase-catalyzed hydrolysis of acetyl-L3-thiocholine (L = 1H or 2H). The isotope effect on V/K is inverse, D3V/K = 0.93 +/- 0.03, which is consistent with conversion of the sp2 hybridized carbonyl carbon of the scissile ester bond of the E + A reactant state to a quasi-tetrahedral structure in the acylation transition state. In contrast, the isotope effect on Vmax under conditions of substrate activation is markedly normal, D3(betaVmax) = 1.29 +/- 0.06, an observation that is consistent with accumulation of a tetrahedral intermediate as the reactant state for catalytic turnover. Generally, tetrahedral intermediates for nonenzymatic ester hydrolyses are high-energy steady-state intermediates. Apparently, butyrylcholinesterase displays an unusual ability to stabilize such intermediates. Hence, the catalytic power of cholinesterases can largely be understood in terms of their ability to stabilize tetrahedral intermediates in the multistep reaction mechanism.

Poly(benzyl ether) dendrimers with strongly fluorescent distyrylbenzene cores as the fluorophores for peroxyoxalate chemiluminescence: insulating effect of dendritic structures on fluorescent sites

Poly(benzyl ether) dendrimers containing strongly fluorescent distyrylbenzene cores were synthesized, and their fluorescence and electrochemical properties as well as the action as fluorophores in the chemiluminescence reactions were investigated. While all the dendrimers exhibited almost the same properties except for their intensities, a characteristic feature due to the dendritic structure was observed in the electrochemical behaviors. In the peroxyoxalate chemiluminescence reactions using these dendrimers as fluorophores, a bimolecular interaction between the high-energy intermediates and fluorophores was established, and a decrease in the chemiluminescence intensity with an increasing generation was observed, which was connected with the insulating effect of the dendritic structures on the core.

An Obligatory Intermediate in the Folding Pathway of Cytochromec552 from Hydrogenobacterthermophilus

The folding mechanism of many proteins involves the population of partially organized structures en route to the native state. Identification and characterization of these intermediates is particularly difficult, as they are often only transiently populated and may play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. Following different spectroscopic probes, and employing state-of-the-art kinetic analysis, we present evidence that the folding mechanism of the thermostable cytochrome c552 from Hydrogenobacter thermophilus does involve the presence of an elusive, yet compact, on-pathway intermediate. Characterization of the folding mechanism of this cytochrome c is particularly interesting for the purpose of comparative folding studies, because H. thermophilus cytochrome c552 shares high sequence identity and structural homology with its homologue from the mesophilic bacterium Pseudomonas aeruginosa cytochrome c551, which refolds through a broad energy barrier without the accumulation of intermediates. Analysis of the folding kinetics and correlation with the three-dimensional structure add new evidence for the validity of a consensus folding mechanism in the cytochrome c family.

The Acetate Switch

To succeed, many cells must alternate between life-styles that permit rapid growth in the presence of abundant nutrients and ones that enhance survival in the absence of those nutrients. One such change in life-style, the "acetate switch," occurs as cells deplete their environment of acetate-producing carbon sources and begin to rely on their ability to scavenge for acetate. This review explains why, when, and how cells excrete or dissimilate acetate. The central components of the "switch" (phosphotransacetylase [PTA], acetate kinase [ACK], and AMP-forming acetyl coenzyme A synthetase [AMP-ACS]) and the behavior of cells that lack these components are introduced. Acetyl phosphate (acetyl approximately P), the high-energy intermediate of acetate dissimilation, is discussed, and conditions that influence its intracellular concentration are described. Evidence is provided that acetyl approximately P influences cellular processes from organelle biogenesis to cell cycle regulation and from biofilm development to pathogenesis. The merits of each mechanism proposed to explain the interaction of acetyl approximately P with two-component signal transduction pathways are addressed. A short list of enzymes that generate acetyl approximately P by PTA-ACKA-independent mechanisms is introduced and discussed briefly. Attention is then directed to the mechanisms used by cells to "flip the switch," the induction and activation of the acetate-scavenging AMP-ACS. First, evidence is presented that nucleoid proteins orchestrate a progression of distinct nucleoprotein complexes to ensure proper transcription of its gene. Next, the way in which cells regulate AMP-ACS activity through reversible acetylation is described. Finally, the "acetate switch" as it exists in selected eubacteria, archaea, and eukaryotes, including humans, is described.

Competitive Inhibitors of Mycobacterium tuberculosis Ribose-5-phosphate Isomerase B Reveal New Information about the Reaction Mechanism

Ribose-5-phosphate isomerase (Rpi), an important enzyme in the pentose phosphate pathway, catalyzes the interconversion of ribulose 5-phosphate and ribose 5-phosphate. Two unrelated isomerases have been identified, RpiA and RpiB, with different structures and active site residues. The reaction catalyzed by both enzymes is thought to proceed via a high energy enediolate intermediate, by analogy to other carbohydrate isomerases. Here we present studies of RpiB from Mycobacterium tuberculosis together with small molecules designed to resemble the enediolate intermediate. The relative affinities of these inhibitors for RpiB have a different pattern than that observed previously for the RpiA from spinach. X-ray structures of RpiB in complex with the inhibitors 4-phospho-d-erythronohydroxamic acid (K(m) 57 microm) and 4-phospho-d-erythronate (K(i) 1.7 mm) refined to resolutions of 2.1 and 2.2 A, respectively, allowed us to assign roles for most active site residues. These results, combined with docking of the substrates in the position of the most effective inhibitor, now allow us to outline the reaction mechanism for RpiBs. Both enzymes have residues that can catalyze opening of the furanose ring of the ribose 5-phosphate and so can improve the efficiency of the reaction. Both enzymes also have an acidic residue that acts as a base in the isomerization step. A lysine residue in RpiAs provides for more efficient stabilization of the intermediate than the corresponding uncharged groups of RpiBs; this same feature lies behind the more efficient binding of RpiA to 4-phospho-d-erythronate.

Computational study on the addition of HCN to methanimine catalyzed by formamidine and formamide

In this paper, we have carried out a theoretical study on the addition of HCN to methanimine with formamidine or formamide using second-order Moller–Plesset perturbation (MP2) method with 6-31+G(d,p) basis sets. At MP2 level, a high-energy intermediate has been located for each pathway. The addition of HCN to methanimine with formamidine has the lowest free energy barrier according to the calculations at MP2 level.

Flow and evaporation cells for the detection of proteins on membranes with the peroxyoxalate chemiluminescent reaction in organic media.

The high-energy intermediates generated in the reaction of bis(2,4,6-trichlorophenyl)oxalate (TCPO) with H2O2 can excite electronically different fluorophores with a high quantum yield in organic solvents. We have previously applied this peroxyoxalate chemiluminescent reaction to the detection of proteins labeled with the fluorescent dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) on polyvinylidene difluoride (PVDF) membranes. In this work, we have investigated the possibility to enhance the sensitivity of this detection method using specially designed cells in which the reagents TCPO and H2O2 in acetone are continuously renewed. In the flow cell, two syringes are used to renew the reagents in the reaction chamber containing the PVDF membrane with blotted proteins labeled with MDPF. In the evaporation cell, a fresh solution of reagents continuously replaces the volume of acetone evaporated in the reaction chamber. Both cells show a low emission background but the observed elution of proteins from the membrane produced by the flow of reagents in acetone limits the maximum sensitivity attainable with these cells. The best result (detection of 1 ng of MDPF-labeled protein) has been obtained with the evaporation cell.

Selective inhibition of Trypanosoma brucei 6-phosphogluconate dehydrogenase by high-energy intermediate and transition-state analogues.

Two series of compounds were designed to mimic the transition state and high-energy intermediates (HEI) of the enzymatic reaction of 6-phosphogluconate dehydrogenase (6PGDH). Sulfoxide analogues (7-11) were designed to mimic the transition state during the oxidation of the substrate to 3-keto-6-phosphogluconate, an enzyme-bound intermediate of the enzyme. Hydroxamate and amide derivatives of d-erythronic acid were designed to mimic the 1,2-cis-enediol HEI of the 6PGDH reaction. These two series of compounds were assayed as competitive inhibitors of the Trypanosoma brucei and sheep liver enzymes, and their selectivity value (ratio sheep/parasite) was calculated. The sulfoxide transition-state analogues showed weak and selective inhibition of the T. brucei enzyme. The hydroxamic derivatives showed potent and selective inhibition of the T. brucei 6PGDH with a Ki in the nanomolar range.

Reaction of Ar2+ with C60 to produce C603+: first observation of double electron-transfer ionization?

The gas-phase reaction of translationally cold Ar2+ (1S0) ions with C60 has been studied using a selected-ion flow tube (SIFT) apparatus. Amongst several product channels detected, which arose from competing reactions of contaminants (N2, O2, H2O) and from the He buffer gas itself as well as from the target process Ar2++C60, we observed an apparently minor but unambiguous signal due to a C603+ product ion. We surmise that this product channel arises from Ar2+(1S0)+C60 in a bimolecular fashion, most probably by an initial (and highly exothermic) direct double electron transfer leading to the generation of either highly excited (C602+)∗ or of electronically excited (Ar)∗. Trication formation from either of these possible high-energy intermediates would then follow according to either an autoionization or a Penning ionization mechanism.

24-Thiacycloartanol, a potent mechanism-based inactivator of plant sterol methyltransferase

The rationally designed substrate mimic of cycloartenol, 24-thiacycloartanol (24-TC) 4A , and its corresponding sulfonium salt 5A were tested against the recombinant sterol methyltransferase (SMT) from Glycine max (soybean). Analog 4A was found to irreversibly inactivate the enzyme generating competitive- and time-dependent inhibition of activity accompanied by a K i value of 2 μM and k inact of 0.3 min −1. Analog 5A , a presumptive high-energy intermediate of the SMT catalyzed reaction, was found to be a reversible, non-competitive-type inhibitor generating a K i value of 55 nM.

Safety vs Efficiency in the Development of a High-Energy Compound

A scalable route to 5-(2-carboxy-pyridin-2-yloxy)-benz[1,2,5]oxadiazole (3) is demonstrated. The synthesis was designed to minimize potential safety issues with a previously practiced route and, in particular, to avoid the handling of 5-hydroxybenzofurazan, which was found to decompose with a large energy release at relatively low temperatures. The new route builds the benzofurazan moiety onto a nicotinonitrile core to avoid high-energy intermediates with low onset temperatures of decomposition.