Synthesis and Antifungal Activity of Norbornene Carboxamide/sulfonamide Derivatives as Potential Fungicides Targeting Laccase.

To explore more potential fungicides with new scaffolds, thirty-seven norbornene carboxamide/sulfonamide derivatives were designed, synthesized, and assayed for inhibitory activity against six plant pathogenic fungi and oomycetes. The preliminary antifungal assay suggested that the title derivatives showed moderate to good antifungal activity against six plant pathogens. Especially, compound 6 e presented excellent in vitro antifungal activity against Sclerotinia sclerotiorum (EC50=0.71 mg/L), which was substantially stronger than pydiflumetofen. In vivo antifungal assay indicated 6 e displayed prominent protective and curative effects on rape leaves infected by S. sclerotiorum. The preliminary mechanism research displayed that 6 e could damage the surface morphology and inhibit the sclerotia formation of S. sclerotiorum. In addition, the in vitro enzyme inhibition bioassay indicated that 6 e displayed pronounced laccase inhibition activity (IC50=0.63 µM), much stronger than positive control cysteine. Molecular docking elucidated the binding modes between 6 e and laccase. The bioassay results and mechanism investigation demonstrated that this class of norbornene carboxamide/sulfonamide derivatives could be promising laccase inhibitors for novel fungicide development.

Inhibitory Potential of New Phenolic Hydrazide-Hydrazones with a Decoy Substrate Fragment towards Laccase from a Phytopathogenic Fungus: SAR and Molecular Docking Studies.

Laccase from pathogenic fungi participates in both the delignification and neutralization of phytoantibiotics. Furthermore, it interferes with the hormone signaling in plants and catalyzes melanization. Infections of these pathogens contribute to loss in forestry, agriculture, and horticulture. As there is still a need to expand knowledge on efficient defense strategies against phytopathogenic fungi, the present study aimed to reveal more information on the molecular mechanisms of laccase inhibition with natural and natural-like carboxylic acid semi-synthetic derivatives. A set of hydrazide-hydrazones derived from carboxylic acids, generally including electron-rich arene units that serve as a decoy substrate, was synthesized and tested with laccase from Trametes versicolor. The classic synthesis of the title inhibitors proceeded with good to almost quantitative yield. Ninety percent of the tested molecules were active in the range of KI = 8-233 µM and showed different types of action. Such magnitude of inhibition constants qualified the hydrazide-hydrazones as strong laccase inhibitors. Molecular docking studies supporting the experimental data explained the selected derivatives' interactions with the enzyme. The results are promising in developing new potential antifungal agents mitigating the damage scale in the plant cultivation, gardening, and horticulture sectors.

Poly(2-oxazoline)s with a 2,2'-Iminodiacetate End Group Inhibit and Stabilize Laccase.

Poly(2-oxazoline)s (POxs) with 2,2'-iminodiacetate (IDA) end groups were investigated as inhibitors for laccase. The polymers with the IDA end groups are reversible, competitive inhibitors for this enzyme. The IC50 values were found to be in a range of 1-3 mm. Compared with IDA alone, the activity was increased by a factor of more than 30; thus indicating that attaching a polymer chain to an inhibitor can already improve the activity of the former. The enzyme activity drops to practically zero upon increasing the concentration of the most active telechelic inhibitor, IDA-PEtOx30 -IDA (PEtOx: poly(2-ethyl-2-oxazoline)), from 5 to 8 mm. This unusual behavior was investigated by means of dynamic light scattering, which showed specific aggregation above 5 mm. Furthermore, the laccase could be stabilized in the presence of POx-IDA, upon addition at a concentration of 20 mm and higher. Whereas laccase becomes completely inactive at room temperature after one week, the stabilized laccase is fully active for at least a month in aqueous solution.

Ecotoxicological test based on inhibition of fungal laccase activity: Application to agrochemicals and the monitoring of pesticide degradation processes.

Ecotoxicological evaluations require the use of assays with several bioindicators from different trophic levels. Only a few ecotoxicological tests using fungi have been developed, reason why, detection of adverse effects from compounds that exert fungicide action may be overlooked. This work developed a toxicity test based on the inhibition of laccase enzymatic activity in the fungus Trametes versicolor. The test was applied to several fungicides and succeeded to determine inhibition values (half maximum effective concentration, EC50) for most of them (flusilazole, imazalil, pyrimethanil, tetraconazole), though a clear dose-response was not evident for others (thiabendazole, metalaxyl). The application on atrazine (herbicide), imidacloprid (insecticide) and oxytetracycline (antibiotic), proved the proposed test is suitable towards other agrochemicals. The test was also used to estimate the detoxification resulting from two different approaches employed in the removal of agrochemicals. (a) First, in the liquid-phase elimination by fungal biomass simultaneously removing atrazine, imazalil, tebuconazole and triadimenol, the test showed a significant decrease in toxicity by biodegradation (adsorption contribution to detoxification was negligible). (b) Second, a solid-phase biomixture (used for pesticide degradation from agricultural wastewater) partially removed atrazine, imazalil, metalaxyl and pyrimethanil after 33 d; nonetheless, this system could not reduce the toxicity of the matrix, and higher laccase inhibition was detected after the treatment. The design test increases the battery of available bioassays to determine the toxicity of agrochemicals, and provides an interesting tool to monitor biodegradation processes.

Synthesis and Structure-Activity Relationship Studies of Hydrazide-Hydrazones as Inhibitors of Laccase from Trametes versicolor.

A series of hydrazide-hydrazones 1-3, the imine derivatives of hydrazides and aldehydes bearing benzene rings, were screened as inhibitors of laccase from Trametes versicolor. Laccase is a copper-containing enzyme which inhibition might prevent or reduce the activity of the plant pathogens that produce it in various biochemical processes. The kinetic and molecular modeling studies were performed and for selected compounds, the docking results were discussed. Seven 4-hydroxybenzhydrazide (4-HBAH) derivatives exhibited micromolar activity Ki = 24-674 µM with the predicted and desirable competitive type of inhibition. The structure-activity relationship (SAR) analysis revealed that a slim salicylic aldehyde framework had a pivotal role in stabilization of the molecules near the substrate docking site. Furthermore, the presence of phenyl and bulky tert-butyl substituents in position 3 in salicylic aldehyde fragment favored strong interaction with the substrate-binding pocket in laccase. Both 3- and 4-HBAH derivatives containing larger 3-tert-butyl-5-methyl- or 3,5-di-tert-butyl-2-hydroxy-benzylidene unit, did not bind to the active site of laccase and, interestingly, acted as non-competitive (Ki = 32.0 µM) or uncompetitive (Ki = 17.9 µM) inhibitors, respectively. From the easily available laccase inhibitors only sodium azide, harmful to environment and non-specific, was over 6 times more active than the above compounds.

Synthesis and Studies of the Inhibitory Effect of Hydroxylated Phenylpropanoids and Biphenols Derivatives on Tyrosinase and Laccase Enzymes.

The impaired activity of tyrosinase and laccase can provoke serious concerns in the life cycles of mammals, insects and microorganisms. Investigation of inhibitors of these two enzymes may lead to the discovery of whitening agents, medicinal products, anti-browning substances and compounds for controlling harmful insects and bacteria. A small collection of novel reversible tyrosinase and laccase inhibitors with a phenylpropanoid and hydroxylated biphenyl core was prepared using naturally occurring compounds and their activity was measured by spectrophotometric and electrochemical assays. Biosensors based on tyrosinase and laccase enzymes were constructed and used to detect the type of protein-ligand interaction and half maximal inhibitory concentration (IC50). Most of the inhibitors showed an IC50 in a range of 20-423 nM for tyrosinase and 23-2619 nM for laccase. Due to the safety concerns of conventional tyrosinase and laccase inhibitors, the viability of the new compounds was assayed on PC12 cells, four of which showed a viability of roughly 80% at 40 µM. In silico studies on the crystal structure of laccase enzyme identified a hydroxylated biphenyl bearing a prenylated chain as the lead structure, which activated strong and effective interactions at the active site of the enzyme. These data were confirmed by in vivo experiments performed on the insect model Tenebrio molitur.

A highly stable laccase from Bacillus subtilis strain R5: gene cloning and characterization.

The gene encoding copper-dependent laccase from Bacillus subtilis strain R5 was cloned and expressed in Escherichia coli. Initially the recombinant protein was produced in insoluble form as inclusion bodies. Successful attempts were made to produce the recombinant protein in soluble and active form. The laccase activity of the recombinant protein was highly dependent on the presence of copper ions in the growth medium and microaerobic conditions during protein production. The purified enzyme exhibited highest activity at 55 °C and pH 7.0. The recombinant protein was highly thermostable, albeit from a mesophilic source, with a half-life of 150 min at 80 °C. Similar to temperature, the recombinant protein was stable in the presence of organic solvents and protein denaturants such as urea. Furthermore, the recombinant protein was successfully utilized for the degradation of various synthetic dyes reflecting its potential use in treatment of wastewater in textile industry. Abbreviations: ABTS,2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid; CBB, Coomassie brilliant blue; SGZ, syringaldazine; DMP, 2,2-dimethoxy phenol.

Molecular Mechanisms Underlying Inhibitory Binding of Alkylimidazolium Ionic Liquids to Laccase.

Water-miscible alkylimidazolium ionic liquids (ILs) are "green" co-solvents for laccase catalysis, but generally inhibit enzyme activity. Here, we present novel insights into inhibition mechanisms by a combination of enzyme kinetics analysis and molecular simulation. Alkylimidazolium cations competitively bound to the TI Cu active pocket in the laccase through hydrophobic interactions. Cations with shorter alkyl chains (C2~C6) entered the channel inside the pocket, exhibiting a high compatibility with laccase (competitive inhibition constant Kic = 3.36~3.83 mM). Under the same conditions, [Omim]Cl (Kic = 2.15 mM) and [Dmim]Cl (Kic = 0.18 mM) with longer alkyl chains bound with Leu296 or Leu297 near the pocket edge and Leu429 around TI Cu, which resulted in stronger inhibition. Complexation with alkylimidazolium cations shifted the pH optima of laccase to the right by 0.5 unit, and might, thereby, lead to invalidation of the Hofmeister series of anions. EtSO4- showed higher biocompatibility than did Ac- or Cl-, probably due to its binding near the TI Cu and its hindering the entry of alkylimidazolium cations. In addition, all tested ILs accelerated the scavenging of 2, 2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, which, however, did not play a determining role in the inhibition of laccase.

Laccase Inhibition by Arsenite/Arsenate: Determination of Inhibition Mechanism and Preliminary Application to a Self-Powered Biosensor.

The reversible inhibition of laccase by arsenite (As(3+)) and arsenate (As(5+)) is reported for the first time. Oxygen-reducing laccase bioelectrodes were found to be inhibited by both arsenic species for direct electron-transfer bioelectrodes (using anthracene functionalities for enzymatic orientation) and for mediated electron-transfer bioelectrodes [using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as an electron mediator]. Both arsenic species were determined to behave via a mixed inhibition model (behaving closely to that of uncompetitive inhibitors) when evaluated spectrophotometrically using ABTS as the electron donor. Finally, laccase bioelectrodes were employed within an enzymatic fuel cell, yielding a self-powered biosensor for arsenite and arsenate. This conceptual self-powered arsenic biosensor demonstrated limits of detection (LODs) of 13 µM for arsenite and 132 µM for arsenate. Further, this device possessed sensitivities of 0.91 ± 0.07 mV/mM for arsenite and 0.98 ± 0.02 mV/mM for arsenate.

Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa.

Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An Arabidopsis double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in Populus trichocarpa. To elucidate the roles of ptr-miR397a and its targets, we characterized the laccase gene family and identified 49 laccase gene models, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all nine transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an ∼40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up graphic Gaussian model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome-based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases, and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.

Halide binding and inhibition of laccase copper clusters: the role of reorganization energy.

Laccase-like proteins are multicopper oxidases involved in several biological and industrial processes. Their application is commonly limited due to inhibition by fluoride and chloride, and as-isolated proteins are often substantially activated by heat, suggesting that multiple redox states can complicate characterization. Understanding these processes at the molecular level is thus desirable but theoretically unexplored. This paper reports systematic calculations of geometries, reorganization energies, and ionization energies for all partly oxidized states of the trinuclear copper clusters in realistic models with ∼200 atoms. Corrections for scalar-relativistic effects, dispersion, and thermal effects were estimated. Fluoride, chloride, hydroxide, or water was bound to the T2 copper site of the oxidized resting state, and the peroxo intermediate was also computed for reference. Antiferromagnetic coupling, assigned oxidation states, and general structures were consistent with known spectroscopic data. The computations show that (i) ligands bound to the T2 site substantially increase the reorganization energy of the second reduction of the resting state and reduce the redox potentials, providing a possible mechanism for inhibition; (ii) the reorganization energy is particularly large for F(-) but also high for Cl(-), consistent with the experimental tendency of inhibition; (iii) reduction leads to release of Cl(-) from the T2 site, suggesting a mechanism for heat/reduction activation of laccases by dissociation of inhibiting halides or hydroxide from T2.

Poly(N-vinylpyrrolidone)-poly(dimethylsiloxane)-based polymersome nanoreactors for laccase-catalyzed biotransformations.

Laccases (Lac) are oxidizing enzymes with a broad range of applications, for example, in soil remediation, as bleaching agent in the textile industry, and for cosmetics. Protecting the enzyme against degradation and inhibition is of great importance for many of these applications. Polymer vesicles (polymersomes) from poly(N-vinylpyrrolidone)-block-poly(dimethylsiloxane)-block-poly(N-vinylpyrrolidone) (PNVP-b-PDMS-b-PNVP) triblock copolymers were prepared and investigated as intrinsically semipermeable nanoreactors for Lac. The block copolymers allow oxygen to enter and reactive oxygen species (ROS) to leave the polymersomes. EPR spectroscopy proved that Lac can generate ROS. They could diffuse out of the polymersome and oxidize an aromatic substrate outside the vesicles. Michaelis-Menten constants Km between 60 and 143 µM and turn over numbers kcat of 0.11 to 0.18 s(-1) were determined for Lac in the nanoreactors. The molecular weight and the PDMS-to-PNVP ratio of the block copolymers influenced these apparent Michaelis-Menten parameters. Encapsulation of Lac in the polymersomes significantly protected the enzyme against enzymatic degradation and against small inhibitors: proteinase K caused 90% less degradation and the inhibitor sodium azide did not affect the enzyme's activity. Therefore, these polymer nanoreactors are an effective means to stabilize laccase.

Highly stable laccase from repeated-batch culture of Funalia trogii ATCC 200800.

The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55-90 degrees C, which was maximal at 80-95 degrees C. It was highly stable within the temperature intervals 20-50 degrees C. The half life of the enzyme was about 2 h and 5 min at 60 degrees C and 70 degrees C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0-9.0. NaN3 and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested. The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications.

A 96-well electrochemical method for the screening of enzymatic activities.

The rapid electrochemical screening of enzyme activities in bioelectronics is still a challenging issue. In order to solve this problem, we propose to use a 96-well electrochemical assay. This system is composed of 96 screen-printed electrodes on a printed circuit board adapted from a commercial system (carbon is used as the working electrode and silver chloride as the counter/reference electrode). The associated device allows for the measurements on the 96 electrodes to be performed within a few seconds. In this work, we demonstrate the validity of the screening method with the commercial laccase from the fungus Trametes versicolor. The signal-to-noise ratio (S/N) is found to be the best way to analyze the electrochemical signals. The S/N follows a saturation-like mechanism with a dynamic linear range of two decades ranging from 0.5 to 75 ng of laccase (corresponding to enzymatic activities from 62 × 10(-6) to 9.37 × 10(-3) µmol min(-1)) and a sensitivity of 3027 µg(-1) at +100 mV versus Ag/AgCl. Laccase inhibitors (azide and fluoride anions), pH optima, and interfering molecules could also be identified within a few minutes.

Purification and characterization of a novel laccase from Coprinus cinereus and decolorization of different chemically dyes.

Laccase is a blue copper oxidase with multiple copper ions and widely distributed in higher plant and fungi. To date, numerous fungal laccases have been reported by many researchers. In present work, a new laccase gene, named CcLCC5I, from Coprinus cinereus was synthesized chemically according to the yeast bias codon and integrated into Pichia pastoris GS115 genome by electroporation. SDS-PAGE analysis showed that the recombinant laccase has a molecular mass of approximately 56.8 kDa. Its biochemical properties was carried out using substrate 2-2(')-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was showed that the optimum pH and temperature of the laccase is 3.0 and 55 °C, respectively. Except for copper ions, most metal ions inhibited the laccase activity at a high concentration about 10 mM. Sodium sulfite can also highly inhibit laccase activity whereas EDTA had no inhibitory effect on the laccase activity. The CcLCC5I have high ability to decolor not only azo but also aryl methane dyes. The recombinant laccase decolored 44.6 % orange G, 54.8 % Crystal Violet, and 87.2 % Malachite green at about 2.6 h. The novel laccase may be a good candidate for breeding engineering strains used in the treatment of industrial effluent containing azo and aryl methane dyes.

A mechanism for NaCl inhibition of Reactive Blue 19 decolorization and ABTS oxidation by laccase.

Laccases produced by white rot fungi have been extensively evaluated for their potential to decolorize textile wastewaters which contain salts like sodium chloride and sodium sulfate. The effect of sodium chloride and sodium sulfate on Trametes versicolor laccase during the decolorization of an anthraquinone dye (Reactive Blue 19) and the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were evaluated by steady-state kinetic analysis. The results showed that, while sodium sulfate did not affect laccase activity, sodium chloride inhibited both ABTS oxidation and dye decolorization. However, the type of inhibition was substrate-dependent: it was hyperbolic, noncompetitive with ABTS and parabolic, noncompetitive with Reactive Blue 19. Furthermore, the results suggested that two chlorides may bind to laccase in the presence of the dye unlike recent inhibition models which suggest that there is only one inhibition site. This investigation is the first to provide evidence for and to propose a two-site model of laccase inhibition, providing new insight into NaCl inhibition of laccase. The proposed model is also useful to predict decolorization rates in the presence of sodium chloride and to determine operating conditions that will minimize inhibition.

Characterization of laccase activity produced by Cryptococcus albidus.

This study deals with the characterization of laccase enzyme activity produced by Cryptococcus albidus. Industrial wastes like effluent and sludge are complex mixtures of a number of chemicals. These chemicals can interfere with the proper functioning of the enzymes used for bioremediation. Thus, it is important to study the effect of such interfering solvents, detergents, metal chelators, and other chemicals on enzyme activity before industrial applications. Laccase showed maximum activity at pH 2.5 and temperature 20-30°C when ABTS was used as a substrate. The enzyme followed Michaelis-Menten kinetics: K(m) was 0.8158 mM and V(max) was 1527.74 U/mg. Laccase showed good thermostability with a half-life of 81 min at 25°C, 77 min at 35°C, 64 min at 45°C, 36 min at 55°C, and 21 min at 65°C. There was no effect of sodium dodceyl sulfate (SDS) (0.1-1.0%) and EDTA (0.1-0.5%) on laccase activity. Sodium azide and 2-mercaptoethanol showed complete inhibition of laccase activity at 0.1% concentration. At lower concentrations of acetone and acetonitrile, laccase was able to maintain its activity. However, the activity was completely inhibited at a concentration of 50% or above of acetone, methanol, 1,4-dioxan, and acetonitrile.

Biochemical characterization of laccase from hairy root culture of Brassica juncea L. and role of redox mediators to enhance its potential for the decolorization of textile dyes.

In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve enhanced decolorization, 2, 2'-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient. Laccase activity of 4.5 U mg(-1) of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg(-1) specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C, respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0-6.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol and L: -cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using hairy roots.

Ptilomycalin A inhibits laccase and melanization in Cryptococcus neoformans.

The antifungal spirocyclic guanidine alkaloid, ptilomycalin A, from marine sponge Monanchora arbuscula, inhibits melanogenesis of Cryptococcus neoformans in vitro through inhibition of biosynthesis of laccase in the melanin biosynthetic pathway with an IC(50) of 7.3 µM.

Extracellular laccase produced by an edible basidiomycetous mushroom, Grifola frondosa: purification and characterization.

A major laccase isozyme (Lac 1) was isolated from the culture fluid of an edible basidiomycetous mushroom, Grifola frondosa. Lac 1 was revealed to be a monomeric protein with a molecular mass of 71 kDa. The N-terminal amino acid sequence of Lac 1 was highly similar to those of laccases of some other white-rot basidiomycetes. Lac 1 showed the typical absorption spectrum of a copper-containing enzyme. The enzyme was stable in a wide pH range (4.0 to 10.0), and lost no activity up to 60 °C for 60 min. The optimal pH of the enzyme activity varied among substrates. The K(m) values of Lac 1 toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dimethoxyphenol, guaiacol, catechol, and 3,4-dihydroxy-L-phenylalanine were 0.0137 mM, 0.608 mM, 0.531 mM, 2.51 mM, and 0.149 mM respectively. Lac 1 activity was remarkably inhibited by the chloride ion, in a reversible manner. Lac 1 activity was also inhibited by thiol compounds.