Physical methods to determine the binding mode of putative ligands for hepatitis C virus NS3 helicase

Several small molecules identified by high-throughput screening (HTS) were evaluated for their ability to bind to a nonstructural protein 3 (NS3) helicase from hepatitis C virus (HCV). Equilibrium dissociation constants (K(d)'s) of the compounds for this helicase were determined using several techniques including an assay measuring the kinetics of isothermal enzyme denaturation at several concentrations of the test molecule. Effects of two nonhydrolyzable ATP analogs on helicase denaturation were measured as controls using the isothermal denaturation (ITD) assay. Two compounds, 4-(2,4-dimethylphenyl)-2,7,8-trimethyl-4,5-quinolinediamine and 2-phenyl-N-(5-piperazin-1-ylpentyl)quinazolin-4-amine, were identified from screening that inhibited the enzyme and had low micromolar dissociation constants for NS3 helicase in the ITD assay. Low micromolar affinity of the quinolinediamine to helicase was also confirmed by nuclear magnetic resonance experiments. Unfortunately, isothermal titration calorimetry (ITC) experiments indicated that a more water-soluble analog bound to the 47/23-mer oligonucleotide helicase substrate with low micromolar affinity as did the substituted quinazolinamine. There was no further interest in these templates as helicase inhibitors due to the nonspecific binding to enzyme and substrate. A combination of physical methods was required to discern the mode of action of compounds identified by HTS and remove undesirable lead templates from further consideration.     

Inactivation of monomeric sarcosine oxidase by reaction with N-(cyclopropyl)glycine

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound flavin adenine dinucleotide (FAD). The present study demonstrates that N-(cyclopropyl)glycine (CPG) is a mechanism-based inhibitor. CPG forms a charge transfer complex with MSOX that reacts under aerobic conditions to yield a covalently modified, reduced flavin (lambda(max) = 422 nm, epsilon(422) = 3.9 mM(-1) cm(-1)), accompanied by a loss of enzyme activity. The CPG-modified flavin is converted at an 8-fold slower rate to 1,5-dihydro-FAD (EFADH(2)), which reacts rapidly with oxygen to regenerate unmodified, oxidized enzyme. As a result, CPG-modified MSOX reaches a CPG-dependent steady-state concentration under aerobic conditions and reverts back to unmodified enzyme upon removal of excess reagent. No loss of activity is observed under anaerobic conditions where EFADH(2) is formed in a reaction that goes to completion at low CPG concentrations. Aerobic denaturation of CPG-modified enzyme yields unmodified, oxidized flavin at a rate similar to the anaerobic denaturation reaction, which yields 1,5-dihydro-FAD. The CPG-modified flavin can be reduced with borohydride, a reaction that blocks conversion to unmodified flavin upon removal of excess CPG or enzyme denaturation. The possible chemical mechanism of inactivation and structure of the CPG-modified flavin are discussed.     

Conformational changes of maize and wheat NADP-malic enzyme studied by quenching of protein native fluorescence

Quenching of tryptophan fluorescence of maize and wheat NADP-malic enzyme by KI and acrylamide was studied after denaturating proteins with guanidine hydrochloride, and subjecting them to different pH values or temperatures. Protein unfolding by guanidine hydrochloride resulted in a red shift of the fluorescence spectrum, providing further support for the motion that several of the tryptophan residues evolved from an apolar to a polar environment. Protein denaturation was accompanied by an increase in the effective dynamic quenching constant values and by loss of the enzyme's activities. Thermal denaturation gave results consistent with the ones observed for chemical denaturation suggesting that a putative intermediate is involved in the denaturation process. Finally, exposure of both enzymes at various pH values allowed us to infer the number of accessible tryptophan residues in the different oligomeric conformations. The results suggest that the aggregation process seems to be different for each enzyme. Thus, as the maize enzyme associated from monomer to tetramer, one tryptophan residue would change from a polar to an apolar environment, while the association of the wheat enzyme would cause that two tryptophan residues to be excluded from quenching. Hitherto, quenching of the tryptophan fluorescence provides a good tool for studying conformational changes of proteins. The future availability of the crystal structures of plant NADP-malic enzymes will offer a good validation point for our model and the technology used.     

Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fischeri NAD(P)H:FMN-oxidoreductase.

The bioluminescent bacterial enzyme system NAD(P)H:FMN-oxidoreductase-luciferase has been used as a test system for ecological monitoring. One of the modes to quench bioluminescence is the interaction of xenobiotics with the enzymes, which inhibit their activity. The use of endogenous flavin fluorescence for investigation of the interactions of non-fluorescent compounds with the bacterial luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri has been proposed. Fluorescence spectroscopy methods have been used to study characteristics of endogenous flavin fluorescence (fluorophore lifetime, the rotational correlation time). The fluorescence anisotropy behaviour of FMN has been analysed and compared to that of the enzyme-bound flavin. The fluorescence characteristics of endogenous flavin of luciferase and NAD(P)H:FMN-oxidoreductase have been shown to be applicable in studying enzymes' interactions with non-fluorescent compounds.     

Effects of paraoxon and ethylparathion on choline oxidase from Alcaligenes species: inhibition and denaturation

The kinetics and thermodynamics of the effects of paraoxon (POX) and ethylparathion (EPA) on choline oxidase (ChOx) were studied. Lineweaver–Burk plots of initial velocity data showed a parallel pattern indicating uncompetitive inhibition versus choline. The inhibition constant (K_I) obtained from the secondary plots for POX and EPA were 0.14 ± 0.01 and 0.48 ± 0.05 mM, respectively, suggesting that POX is a more potent inhibitor of ChOx than EPA. UV absorption was used to monitor the denaturation of ChOx by POX and EPA. A decrease in FAD fluorescence associated with the interaction of POX and EPA with ChOx suggested a tertiary structural change. Interaction of the enzyme molecule with POX or EPA resulted in inhibition and subsequently denaturation of the enzyme. The results indicate that inhibition and denaturation of the enzyme by POX and EPA are linked, but not parallel events, with inhibition occurring at lower concentrations with respect to denaturation. This suggests that the loss of initial velocity of the enzyme is an active site specific effect and not due to global conformational changes induced by the inhibitors.     

Screening and molecular dynamics simulation of compounds inhibiting MurB enzyme of drug-resistant Mycobacterium tuberculosis: An in-silico approach

Mycobacterium tuberculosis (MTB) is becoming more and more resistant to drugs and it is a common problem, making current antimicrobials ineffective and highlighting the need for new TB drugs. One of the promising targets for treating MTB is MurB enzymes. This study aimed to identify potential inhibitors of MurB enzymes in M. tuberculosis, as drug resistance among MTB is a significant problem. Attempts are being made to conduct a virtual screening of 30,417 compounds, and thirty-two compounds were chosen for further analysis based on their binding conformations. The selected compounds were assessed for their drug-likeness, pharmacokinetics, and physiochemical characteristics, and seven compounds with binding energy lower than flavin (FAD) were identified. Further, molecular dynamics simulation analysis of these seven compounds found that four of them, namely DB12983, DB15688, ZINC084726167, and ZINC254071113 formed stable complexes with the MurB binding site, exhibiting promising inhibitory activity. These compounds have not been mentioned in any other study, indicating their novelty. The study suggests that these four compounds could be promising candidates for treating MTB, but their effectiveness needs to be validated through in vitro and in vivo experiments. Overall, the findings of this study provide new insight into potential drug targets and candidates for combating drug-resistant MTB.     

The dissociation of flavin coenzymes from trypsin-solubilized NADPH/Cytochrome c (P-450) reductase of pig-liver microsomes.

The change in fluorescence emission at 520 nm after excitation at 365 nm was used to investigate the effect of pH and ionic strength on the dissociation of flavin cofactors from microsomal NADPH/cytochrome c (P-450) reductase. In the unmodified enzyme both the FAD and FMN moieties appeared to dissociate at a similar rate and followed first-order kinetics. The rate constant for the dissociation was increased by low pH and high ionic strength, particularly in the range pH 4.4-3.8 (0.02 M acetate buffer) where the rate constants increased 80-fold. Modification of the enzyme by treatment with p-chloromercuribenzoate enhanced the rate of flavin dissociation and, in the region of pH 4, resulted in a biphasic increase in fluorescence consistent with two simultaneous parallel first-order dissociations. It was concluded that p-chloromercuribenzoate treatment modified the protein so that the two flavin cofactors dissociated at different rates. Using the measured rate constants for the dissociations, and the known variation in fluorescence of flavin nucleotides with pH, an analogue computer simulation of the dissociation as well as a manual curve-fitting procedure showed that the biphasic response could be explained as a simultaneous rapid dissociation of FAD and a slower loss of FMN from the protein.     

Transient model of thermal deactivation of enzymes

The kinetics of enzyme deactivation provide useful insights on processes that determine the level of biological function of any enzyme. Photinus pyralis (firefly) luciferase is a convenient enzyme system for studying mechanisms and kinetics of enzyme deactivation, refolding, and denaturation caused by various external factors, physical or chemical by nature. In this report we present a study of luciferase deactivation caused by increased temperature (i.e., thermal deactivation). We found that deactivation occurs through a reversible intermediate state and can be described by a Transient model that includes active and reversibly inactive states. The model can be used as a general framework for analysis of complex, multiexponential transient kinetics that can be observed for some enzymes by reaction progression assays. In this study the Transient model has been used to develop an analytical model for studying a time course of luciferase deactivation. The model might be applicable toward enzymes in general and can be used to determine if the enzyme exposed to external factors, physical or chemical by nature, undergoes structural transformation consistent with thermal mechanisms of deactivation.     

The ligand affinity of proteins measured by isothermal denaturation kinetics

An isothermal denaturation kinetic method was developed for identifying potential ligands of proteins and measuring their affinity. The method is suitable for finding ligands specific toward proteins of unknown function and for large-scale drug screening. It consists of analyzing the kinetics of isothermal denaturation of the protein-with and without the presence of potential specific ligands-as measured by long-wavelength fluorescent dyes whose quantum yield increases when bound to hydrophobic regions exposed upon unfolding of the proteins. The experimental procedure was developed using thymidylate kinase and stromelysin as target proteins. The kinetics of thermal unfolding of both of these enzymes were consistent with a pathway of two consecutive first-order rate-limiting steps. Reflecting the stabilizing effect of protein/ligand complexes, the presence of specific ligands decreased the value of the rate constants of both steps in a dose-dependent manner. The dependence of the rate constants on ligand concentration obeyed a simple binding isotherm, the analysis of which yielded an accurate equilibrium constant for ligand binding. The method was validated by comparing its results with those obtained under the same conditions by steady-state fluorescence spectroscopy, circular dichroism, and uv spectrophotometry: The corresponding rate constants were comparable for each of the analytical detection methods.     

Riboflavin analogs and inhibitors of riboflavin biosynthesis

Flavins are active components of many enzymes. In most cases, riboflavin (vitamin B₂) as a coenzyme represents the catalytic part of the holoenzyme. Riboflavin is an amphiphatic molecule and allows a large variety of different interactions with the enzyme itself and also with the substrate. A great number of active riboflavin analogs can readily be synthesized by chemical methods and, thus, a large number of possible inhibitors for many different enzyme targets is conceivable. As mammalian and especially human biochemistry depends on flavins as well, the target of the inhibiting flavin analog has to be carefully selected to avoid unwanted effects. In addition to flavoproteins, enzymes, which are involved in the biosynthesis of flavins, are possible targets for anti-infectives. Only a few flavin analogs or inhibitors of flavin biosynthesis have been subjected to detailed studies to evaluate their biological activity. Nevertheless, flavin analogs certainly have the potential to serve as basic structures for the development of novel anti-infectives and it is possible that, in the future, the urgent need for new molecules to fight multiresistant microorganisms will be met.     

Escherichia coli DNA photolyase is a flavoprotein

Escherichia coli DNA photolyase (photoreactivating enzyme) was purified to homogeneity from a strain that greatly overproduces the protein. The purified enzyme has absorption peaks at 280 and 380 nm, a fluorescence emission peak at 480 nm and, upon denaturation, releases a chromophore that has the spectroscopic properties of flavin adenine dinucleotide (FAD), indicating that FAD is an intrinsic chromophore of the enzyme.     

Combinatorial enzymatic assay for the screening of a new class of bacterial cell wall inhibitors

We have developed a screening assay by thin-layer chromatography (TLC) to identify inhibitors for the bacterial essential enzymes MurA, -B, and -C. Libraries of compounds were synthesized using the mix-and-split combinatorial chemistry approach. Screening of the pooled compounds using the developed assay revealed the presence of many pools active in vitro. Pools of interest were tested for antibacterial activity. Individual molecules in the active pools were synthesized and retested with the TLC assay and with bacteria. We focused on the best five compounds for further analysis. They were tested for inhibition on each of the three enzymes separately, and showed no inhibition of MurA or MurB activity but were all inhibitors of MurC enzyme. This approach yielded interesting lead compounds for the development of novel antibacterial agents.     

Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf). Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Galf) is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the flavin must be in the reduced state for activity and the exact role of the flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that NADPH is an effective redox partner of TcUGM. The substrate, UDP-galactopyranose, protects the enzyme from reacting with molecular oxygen allowing TcUGM to turnover ～1000 times for every NADPH oxidized. Spectral changes consistent with a flavin iminium ion, without the formation of a flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the flavin acting as a nucleophile. In support of this role, a flavin-galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented.     

Picomolar analysis of flavins in biological samples by dynamic pH junction-sweeping capillary electrophoresis with laser-induced fluorescence detection

Sensitive capillary electrophoresis (CE) methods are required for emerging areas of biochemical research such as the metabolome. In this report, dynamic pH junction-sweeping CE with laser-induced fluorescence (LIF) detection is applied as a robust single method to analyze trace amounts of three flavin derivatives, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), from several types of samples including bacterial cell extracts, recombinant protein, and biological fluids. Submicromolar amounts of flavin coenzymes were measured directly from formic acid cell extracts of Bacillus subtilis. Significant differences in flavin concentration were measured in cell extracts derived from either glucose or malate as the carbon source in the culture media. Quantitative assessment of FAD and FMN content from selected flavoenzymes was demonstrated after heat denaturation to release noncovalently bound coenzymes and deproteinization. This method was also applied to the analysis of free flavins in pooled human plasma and urine without the need for laborious off-line sample preconcentration. Picomolar detectability of flavins by CE-LIF detection was realized with on-line preconcentration (up to 15% capillary length used for injection) by dynamic pH junction-sweeping, resulting in a limit of detection (S/N = 3) of about 4.0 pM for FAD and FMN. This represents over a 60-fold improvement in concentration sensitivity compared to those of previous techniques using conventional injections. The method was validated in terms of reproducibility, sensitivity, linearity, and specificity. Flavin analysis by dynamic pH junction-sweeping CE-LIF offers a simple, yet sensitive way to analyze trace levels of flavin metabolites from complex biological samples.     

Kinetics of Acrylodan-Labelled cAMP-Dependent Protein Kinase Catalytic Subunit Denaturation

Fluorescence spectroscopy was used to study denaturation of cAMP-dependent protein kinase catalytic subunit labeled with an acrylodan moiety. The dye was covalently bound to a cystein residue introduced into the enzyme by replacement of arginine in position 326 in the native sequence, located near the enzyme active center. This labeling had no effect on catalytic activity of the enzyme, but provided possibility to monitor changes in protein structure through measuring the fluorescence spectrum of the dye, which is sensitive to changes in its environment. This method was used to monitor denaturation of the protein kinase catalytic subunit and study the kinetics of this process as well as influence of specific ligands on stability of the protein. Stabilization of the enzyme structure was observed in the presence of adenosine triphosphate, peptide substrate RRYSV and inhibitor peptide PKI[5-24].     

From time temperature integrator kinetics to time temperature integrator tolerance levels: heat-treated milk

Six milk compounds were studied as potential intrinsic time temperature integrators (TTIs) for the assessment of heat-treated milk. These include the enzymes alkaline phosphatase and lactoperoxidase, the whey protein beta-lactoglobulin and the chemical compounds hydroxymethylfurfural, lactulose and furosine. In previous research the inactivation/denaturation/formation kinetics of these compounds were analyzed under isothermal and nonisothermal conditions and evaluated for variability of the milk composition. The present paper focuses on the implementation of the TTIs. TTIs are validated with respect to microbiological indices and quality attributes, and a quantitative relationship between the denaturation, inactivation or formation of the TTIs and technological processes is established by construction of general time temperature tolerance (TTT) diagrams. In these diagrams temperature time combinations are presented, which lead to the same formation, inactivation or denaturation of TTIs, or result in the same level of microbiological destruction or quality degradation of the product. TTT-diagrams are very informative since they allow visualization of the impact of a thermal process on milk and evaluation of criteria for evaluating milk authenticity (conformity of the product with the terminology applied). Moreover, the optimum combination of temperature and time of heating may be readily deduced from these diagrams.     

Real-time enzyme dynamics illustrated with fluorescence spectroscopy of p-hydroxybenzoate hydroxylase

We have used the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) to illustrate that a strongly fluorescent donor label can communicate with the flavin via single-pair F?rster resonance energy transfer (spFRET). The accessible Cys-116 of PHBH was labeled with two different fluorescent maleimides with full preservation of enzymatic activity. One of these labels shows overlap between its fluorescence spectrum and the absorption spectrum of the FAD prosthetic group in the oxidized state, while the other fluorescent probe does not have this spectral overlap. The spectral overlap strongly diminished when the flavin becomes reduced during catalysis. The donor fluorescence properties can then be used as a sensitive antenna for the flavin redox state. Time-resolved fluorescence experiments on ensembles of labeled PHBH molecules were carried out in the absence and presence of enzymatic turnover. Distinct changes in fluorescence decays of spFRET-active PHBH can be observed when the enzyme is performing catalysis using both substrates p-hydroxybenzoate and NADPH. Single-molecule fluorescence correlation spectroscopy on spFRET-active PHBH showed the presence of a relaxation process (relaxation time of 23 micros) that is related to catalysis. In addition, in both labeled PHBH preparations the number of enzyme molecules reversibly increased during enzymatic turnover indicating that the dimer-monomer equilibrium is affected.     

Thermal stability of a flavoprotein assessed from associative analysis of polarized time-resolved fluorescence spectroscopy

Upon gradually heating a particular mutant of the flavoprotein NADH peroxidase, it was found from the peculiar time-resolved fluorescence anisotropy pattern of the flavin prosthetic group (FAD) that, at elevated temperature, FAD is released from the tetrameric enzyme. Since in this case a mixture of free and enzyme-bound FAD contributes to the time-dependent fluorescence anisotropy, its analysis can only be accomplished by an associative fitting model, in which specific fluorescence lifetimes of both species are linked to specific correlation times. In this letter the general approach to the associative polarized fluorescence decay analysis is described. The procedure can be used for other flavoproteins to determine the temperature at which the onset of thermal denaturation will start, leading to release of the flavin prosthetic group. Received: 20 November 1998 / Revised version: 6 April 1999 / Accepted: 8 April 1999     

Single-site oxidation, cysteine 108 to cysteine sulfinic acid, in D-amino acid oxidase from Trigonopsis variabilis and its structural and functional consequences

One of the primary sources of enzyme instability is protein oxidative modification triggering activity loss or denaturation. We show here that the side chain of Cys108 is the main site undergoing stress-induced oxidation in Trigonopsis variabilis d-amino acid oxidase, a flavoenzyme employed industrially for the conversion of cephalosporin C. High-resolution anion-exchange chromatography was used to separate the reduced and oxidized protein forms, which constitute, in a molar ratio of about 3:1, the active biocatalyst isolated from the yeast. Comparative analysis of their tryptic peptides by electrospray tandem mass spectrometry allowed unequivocal assignment of the modification as the oxidation of Cys108 into cysteine sulfinic acid. Cys108 is likely located on a surface-exposed protein region within the flavin adenine dinucleotide (FAD) binding domain, but remote from the active center. Its oxidized side chain was remarkably stable in solution, thus enabling the relative biochemical characterization of native and modified enzyme forms. The oxidation of Cys108 causes a global conformational response that affects the protein environment of the FAD cofactor. In comparison with the native enzyme, it results in a fourfold-decreased specific activity, reflecting a catalytic efficiency for reduction of dioxygen lowered by about the same factor, and a markedly decreased propensity to aggregate under conditions of thermal denaturation. These results open up unprecedented routes for stabilization of the oxidase and underscore the possible significance of protein chemical heterogeneity for biocatalyst function and stability.