Enzymatic and spectroscopic studies on the activation or inhibition effects by substituted phenolic compounds in the oxidation of aryldiamines and catechols catalyzed by Rhus vernicifera laccase

The effect of various phenolic compounds on the activity of Rhus vernicifera laccase (Lc) has been evaluated using two different substrates, N,N-dimethyl-p-phenylenediamine and p-tert-butylcatechol. The observed effect strongly depends on the phenol employed and involves either a moderate activation, by halophenols, or inhibition, by acidic phenols. The collective data are consistent with an open active site in Lc, which is capable of accommodating more than one substrate or phenol molecule. According to NMR relaxation experiments, a phenol molecule binds at an average distance from type 1 Cu of about 6 Å, while evidence from electron paramagnetic resonance (EPR) experiments shows that binding of another phenol molecule induces a change, and probably occurs close to, the type 2/type 3 cluster. The effect of phenolic compounds on Lc reactivity is related to a modification of the substrate affinity for the enzyme. This affinity can either be increased, probably through π-stacking or other types of interactions, or decreased, due to competition for the same site. In addition, the alteration induced in the trinuclear copper cluster has a marked effect on the enzyme reactivity. The inhibition observed with acidic phenols is probably due to the protonation of an enzyme intermediate produced at the trinuclear site, e.g. the peroxy intermediate, that causes the release of hydrogen peroxide and prevents the reaction of this intermediate with the substrate.     

Spectroscopic characterization and O2 reactivity of the trinuclear Cu cluster of mutants of the multicopper oxidase Fet3p

Fet3p is a multicopper oxidase that uses four copper ions (one type 1, one type 2, and one type 3 binuclear site) to couple substrate oxidation to the reduction of O(2) to H(2)O. The type 1 Cu site shuttles electrons between the substrate and the type 2/type 3 Cu sites which form a trinuclear Cu cluster that is the active site for O(2) reduction. This study extends the spectroscopic and reactivity studies that have been conducted with type 1-substituted Hg (T1Hg) laccase to Fet3p and a mutant of Fet3p in which the trinuclear Cu cluster is perturbed. To examine the reaction between the trinuclear Cu cluster and O(2), the type 1 Cu Cys(484) was mutated to Ser, resulting in a type 1-depleted (T1D) form of the enzyme. Additional His to Gln mutations were made at the trinuclear cluster to further probe specific contributions to reactivity. One of these mutants (His(126)Gln) produces the first stable but perturbed trinuclear Cu cluster (T1DT3' Fet3p). Spectroscopic characterization (absorption, circular dichroism, magnetic circular dichroism, and electron paramagnetic resonance) of the resting trinuclear sites in T1D and T1DT3' Fet3p reveal that the His(126)Gln mutation changes the electronic structure of both the type 3 and type 2 Cu sites. The trinuclear clusters in T1D and T1DT3' Fet3p react with O(2) to produce peroxide intermediates analogous to that observed in T1Hg laccase. Spectroscopic data on the peroxide intermediates in the three forms provide further insight into the structure of this intermediate. In T1D Fet3p, the decay of this peroxide intermediate is pH-dependent, and the rate of decay is 10-fold higher at low pH. In T1DT3' Fet3p, the decay of the peroxide intermediate is pH-independent and is slow at all pH's. This change in the pH dependence provides new insight into the mechanism of intermediate decay involving reductive cleavage of the O-O bond.     

Deactivation of cellulases by phenols

Pretreatment of lignocellulosic materials may result in the release of inhibitors and deactivators of cellulose enzyme hydrolysis. We report the identification of phenols with major inhibition and/or deactivation effect on enzymes used for conversion of cellulose to ethanol. The inhibition effects were measured by combining the inhibitors (phenols) with enzyme and substrate immediately at the beginning of the assay. The deactivation effects were determined by pre-incubating phenols with cellulases or β-glucosidases for specified periods of time, prior to the respective enzyme assays. Tannic, gallic, hydroxy-cinnamic, and 4-hydroxybenzoic acids, together with vanillin caused 20-80% deactivation of cellulases and/or β-glucosidases after 24h of pre-incubation while enzymes pre-incubated in buffer alone retained all of their activity. The strength of the inhibition or deactivation effect depended on the type of enzyme, the microorganism from which the enzyme was derived, and the type of phenolic compounds present. β-Glucosidase from Aspergillus niger was the most resistant to inhibition and deactivation, requiring about 5 and 10-fold higher concentrations, respectively, for the same levels of inhibition or deactivation as observed for enzymes from Trichoderma reesei. Of the phenol molecules tested, tannic acid was the single, most damaging aromatic compound that caused both deactivation and reversible loss (inhibition) of all of enzyme activities tested.     

N-Ethylmaleimide inhibition of the catalytic activities of the Dunaliella salina coupling factor 1 (CF1) and the restoration of the inhibition of the CF1 ATPase activity by N-ethylmaleimide

The sensitivity of the catalytic activities of the D. salina chloroplast coupling factor 1 (CF₁) to chemical modification by N-ethylmaleimide has been investigated. (i) When D. salina thylakoid membranes are treated with N-ethylmaleimide, both photophosphorylation and the inducible CF₁ ATPase activity are partially (approx. 60%) inhibited. The inhibition of both activities does not require the presence of a proton-motive force, and the inhibition of photophosphorylation is directly related to the N-ethylmaleimide-covalent modification of CF₁ as shown by (a) the time-course for the inhibition and (b) the maximal extent of inhibition. (ii) Treatment of the purified, latent, D. salina CF₁ with low concentrations of N-ethylmaleimide also results in the partial (approx. 60%) inhibition of the inducible ATPase activity (I₅₀ ≈ 50 μM). The inhibition does not require the presence of the chemical modifier during the activation of the enzyme. (iii) N-ethylmaleimide-induced inhibition of the ATPase activity of either membrane-bound or solubilized CF₁ is partially reversed by either (a) prolonged incubation at low concentrations of N-ethylmaleimide or (b) short incubation times at high concentrations of N-ethylmaleimide. The results are interpreted as indicating multiple binding sites on the D. salina CF₁ that have different rates of reactivity with N-ethylmaleimide. Those sites (or site) that react rapidly with N-ethylmaleimide cause(s) an inhibition of both ATP synthase and ATPase activities, whereas those sites (or site) that react more slowly partially restore(s) the original-ATPase activity. The effects of N-ethylmaleimide on the catalytic activity of D. salina CF₁ are probably mediated by N-ethylmaleimide-induced conformational changes of the enzyme.     

Modulation of cytochrome oxidase kinetics by indirect antibody action

Polyclonal antibodies raised against isolated subunit V from beef heart cytochrome oxidase or against the intact enzyme increase its apparent affinity for the substrate cytochrome c at the high-affinity site while diminishing the turnover at that site. At the low-affinity site the major action of both types of antibody is to reduce the apparent affinity for cytochrome c. At high ionic strengths the kinetic effect of anti-subunit V is very small although it still binds to the enzyme. The results are interpreted in terms of a model for the enzyme in which antibodies can modulate cytochrome oxidase kinetics by affecting the binding of cytochrome c, even if the antibody-binding site is on a subunit not directly involved in substrate binding.     

Horseradish peroxidase in ionic liquids: Reactions with water insoluble phenolic substrates

The reactivity of horseradish peroxidase (HRP) with water insoluble phenolic compounds has been studied in 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF₄])/water mixtures. The enzyme retained some catalytic activity up to 90% ionic liquid in water at 25 °C only at pH values higher than 9.0. Activity steadily decreased towards neutral and acidic conditions, as judged by 4-aminoantypirin/phenol activity tests. Inhibition of horseradish peroxidase under neutral acidic condition was due to the binding of fluoride anions released from tetrafluoroborate anion to the heme iron as demonstrated by the sharp UV–visible absorption transition diagnostic of the conversion from a five coordinated to a six coordinated high spin ferric heme iron. Thus, reactions with water insoluble phenols were carried out under alkaline reaction conditions and 75% [BMIM][BF₄]/water mixture. Under these conditions, the distribution of the reaction products was much narrower with respect to that observed in aqueous buffers or water/dimethylformamide or water/dimethylsulfoxide mixtures, and polymeric species other than dimers were not observed. Technical scale preparations of a novel 4-phenylphenol ortho dimer [2,2′-bi-(4-phenylphenol)] with a high yield of the desired product were obtained.     

Identification of aspartic acid and histidine residues mediating the reaction mechanism and the substrate specificity of the human UDP-glucuronosyltransferases 1A

The human UDP-glucuronosyltransferase UGT1A6 is the primary phenol-metabolizing UDP-glucuronosyltransferase isoform. It catalyzes the nucleophilic attack of phenolic xenobiotics on UDP-glucuronic acid, leading to the formation of water-soluble glucuronides. The catalytic mechanism proposed for this reaction is an acid-base mechanism that involves an aspartic/glutamic acid and/or histidine residue. Here, we investigated the role of 14 highly conserved aspartic/glutamic acid residues over the entire sequence of human UGT1A6 by site-directed mutagenesis. We showed that except for aspartic residues Asp-150 and Asp-488, the substitution of carboxylic residues by alanine led to active mutants but with decreased enzyme activity and lower affinity for acceptor and/or donor substrate. Further analysis including mutation of the corresponding residue in other UGT1A isoforms suggests that Asp-150 plays a major catalytic role. In this report we also identified a single active site residue important for glucuronidation of phenols and carboxylic acid substrates by UGT1A enzyme family. Replacing Pro-40 of UGT1A4 by histidine expanded the glucuronidation activity of the enzyme to phenolic and carboxylic compounds, therefore, leading to UGT1A3-type isoform in terms of substrate specificity. Conversely, when His-40 residue of UGT1A3 was replaced with proline, the substrate specificity shifted toward that of UGT1A4 with loss of glucuronidation of phenolic substrates. Furthermore, mutation of His-39 residue of UGT1A1 (His-40 in UGT1A4) to proline led to loss of glucuronidation of phenols but not of estrogens. This study provides a step forward to better understand the glucuronidation mechanism and substrate recognition, which is invaluable for a better prediction of drug metabolism and toxicity in human.     

Simple phenolic glycosides as potential regulators of the IAA-oxidase system

The effect of phenols and simple phenolic glycosides on the activity of IAA-oxidase isolated from gherkin seedlings was studied in experimentsin vitro. Phenol stimulated the enzyme system activity, eugenol and quinol were proven as inhibitors. Simple phenolic glycosides (arbutin, gein and phenol glucoside) influenced IAA-oxidase activity only if β-glycosidase was present: rree phenols released from their bound form increased or decreased the IAA level. The potential fegulatory effect of simple phenolic glycosides on the IAA level in plants has been discussed; this effect is thought to be mediated by free phenols and by influence on the IAA-oxidase system.     

The roles of veratryl alcohol and nonionic surfactant in the oxidation of phenolic compounds by lignin peroxidase

Veratryl alcohol (VA) at higher concentration stimulated the lignin peroxidase (LiP)-catalyzed oxidation of phenolic compounds remarkably. This novel phenomenon was due to its competition with the phenols for the active site of the enzyme and to the high reactivity of the formed cation radical of VA (VA+*) which resulted in an additional oxidation of the phenols. The influence of the nonionic surfactant Tween 80 on the VA-enhanced LiP-catalyzed oxidation of phenols depended on its concentration. At lower concentration it had a small synergetic effect but at higher concentration it decreased the initial rate. Studies of the capillary electrophoretic behavior of LiP in the presence of Tween 80 showed that this effect was caused by the surfactant aggregation on LiP which, at higher surfactant concentrations, might impede the access of VA to its binding site on LiP and, consequently, the VA+* formation.     

pK values for active site residues of carboxypeptidase A

The phenolic group of active site residue Tyr-248 in carboxypeptidase A has a pKa value of 10.06, as determined from the pH dependence of its rate of nitration by tetranitromethane. The decrease in enzyme activity (kcat/Km) in alkaline solution, characterized by a pKa value of approximately 9.0 (for cobalt carboxypeptidase A), is associated with the protonation state of an imidazole ligand of the active-site metal ion, as indicated by a selective pH dependence of the 1H NMR spectrum of the enzyme. Inhibition of the cobalt-substituted enzyme by 2-(1-carboxy-2-phenylethyl)phenol and its 4,6-dichloro- and 4-phenylazo-derivatives confirms that the decrease in enzyme activity (kcat/Km) in acidic solution, characterized by a pKa value of 5.8, is due to the protonation state of a water molecule bound to the active-site metal ion in the absence of substrate. Changes in the coordination number of the active-site metal ion are seen in its visible absorption spectrum as a consequence of binding of the phenolic inhibitors. Conventional concepts regarding the mechanisms of the enzyme are brought into question.     

NAD-phenol complex formation, the inhibition of malate dehydrogenase by phenols, and the influence of phenol substitutents on inhibitory effectiveness.

Kinetic analyses indicate that inhibition by phenols of the forward reaction of malate dehydrogenase involves the binding of two molecules of phenol. One is bound as phenol, the other as a charge transfer complex of phenol with NAD. Inhibition of the reverse reaction by phenol involves the binding of only a single phenol molecule per active unit of enzyme. Kinetic evidence for this binding pattern is supported by spectral evidence in which ultraviolet absorbance and circular dichroism studies show binding of the NAD-phenol complex by malate dehydrogenase. Circular dichroism difference spectra indicate that phenol alone also binds to malate dehydrogenase.The apparent inhibition constants for fourteen variously substituted phenols were found to be significantly correlated with the hydrophobic binding constant (π), the Hammet σ function and the NAD-phenol charge transfer association constant of the individual phenols. The degree of dependency of the apparent K_i on the hydrophobicity of phenols suggests that the observed inhibition occurs via binding of phenol and/or NAD-phenol complex in hydrophobic regions of the malate dehydrogenase molecule.     

Effect of a charged residue at the 213th site of thermolysin on the enzymatic activity

Considering the electrostatic potential of active site, four mutants of thermolysin (EC 3.4.24.4) are designed in an attempt to change the optimum pH of the hydrolytic activity toward acidic regions. On the basis of the numerical calculation of the electrostatic potential in the thermolysin molecule, Asp213 is targeted to be replaced by a basic residue, His, Lys, Arg or a neutral one, Asn. The mutant enzymes are produced inBacillus subtilis as a host using the method of site-directed mutagenesis and their optimum pH values for hydrolyzing a synthetic substrate furylacryloyl-Gly-l-Leu-NH₂ are found to be lowered by 0.2–0.4 pH units with reference to the wild type enzyme. The pl shifts of the mutants are evaluated. Neither optimum pH nor pl shift can be explained by the contribution of the pK change only at the mutation site. We find a clear negative correlation between the activities at pH 7.0 and the pI values among the four mutants and wild-type enzyme. It suggests that the contribution of pK shift of other residues must be taken into account in order to explain the activity change. Little change of thermal stability is observed among the mutants and wild type enzymes.     

Recognition dynamics of trinuclear copper cluster and associated histidine residues through conserved or semi-conserved water molecules in human Ceruloplasmin: The involvement of aspartic and glutamic acid gates

Human Ceruloplasmin belongs to the family of multi-copper oxidases and it is involved in different physiological processes, copper ion transport, iron metabolism, iron homeostasis, and biogenic amine metabolism. MD-simulation studies have indicated the higher hydrophilic susceptibility of the trinuclear copper cluster in native CP compared to its oxygen bound form. The copper (T2/T3) atom Cu3047 of the cluster, which is close to T1 copper center Cu3052 (~13 Å) has a higher affinity for water molecules compared to other copper centers. The water molecules of W3, W4, W5, W9, and W12 conserved water sites are coordinated to Cu3047, where W3, W9, and W12 centers are found to play some crucial role in the stabilization of native trinuclear copper cluster. The hydrogen bonding interaction of Asp169, Glu112, Asp995, and Glu1032 residues with the copper-bound conserved water molecules (W3, W4, W5, W10, and W11) in native CP is observed to be unique. The conformational flexibility of Asp169 and Glu112 and their association with the copper-bound water molecules, but the absence of such interaction in O₂-bound simulated structure of the enzyme is indicating some plausible rational on the role of these acidic residues in the gating of O₂ molecule in the native trinuclear Cu cluster of CP. The simulation results may shade some new light on the biochemistry/chemistry of CP, specially on the hydration dynamics of the trinuclear copper cluster.     

Antithrombin activity of the hirudin N-terminal core domain residues 1-43

A tetrahedral intermediate is the prominent feature of the generally accepted mechanism for aspartate transcarbamoylase. We have synthesized N-pyrophosphoryl-L-aspartate as a charged analogue of the postulated intermediate. Surprisingly, its affinity for the enzyme from Escherichia coli was substantially lower than that of the previously known inhibitor phosphonoacetyl-L-aspartate which contained a trigonal carbonyl group. Similar results were obtained with the corresponding mercaptosuccinate derivatives. We also tested a number of new pyrophosphate analogues as inhibitors. Our results cast doubt on some aspects of the current model for the mechanism of this enzyme.     

Selective Detoxification of Phenols by Pichia pastoris and Arabidopsis thaliana Heterologously Expressing the PtUGT72B1 from Populus trichocarpa

Phenols are present in the environment and commonly in contact with humans and animals because of their wide applications in many industries. In a previous study, we reported that uridine diphosphate-glucose-dependent glucosyltransferase PtUGT72B1 from Populus trichocarpa has high activity in detoxifying trichlorophenol by conjugating glucose. In this study, more experiments were performed to determine the substrate specificity of PtUGT72B1 towards phenolic compounds. Among seven phenols tested, three were glucosylated by PtUGT72B1 including phenol, hydroquinone, and catechol. Transgenic Arabidopsis plants expressing the enzyme PtUGT72B1 showed higher resistance to hydroquinone and catechol but more sensitivity to phenol than wild type plants. Transgenic Pichia pastoris expressing PtUGT72B1 showed enhanced resistance to all three phenols. Compared with wild type Arabidopsis plants, transgenic Arabidopsis plants showed higher removal efficiencies and exported more glucosides of phenol, phenyl β-D-glucopyranoside, to the medium after cultured with the three phenols. Protein extracts from transgenic Arabidopsis plants showed enhanced conjugating activity towards phenol, hydroquinone and catechol. PtUGT72B1 showed much higher expression level in Pichia pastoris than in Arabidopsis plants. Kinetic analysis of the PtUGT72B1 was also performed.     

Mechanistic features of lignin peroxidase-catalyzed oxidation of substituted phenols and 1,2-dimethoxyarenes

The steady state kinetic parameters Km and kcat for the oxidation of phenolic substrates by lignin peroxidase correlated with the presteady state kinetic parameters Kd and k for the reaction of the enzyme intermediate compound II with the substrates, indicating that the latter is the rate-limiting step in the catalytic cycle. ln Km and ln Kd values for phenolic substrates correlated with redox properties, unlike ln kcat and ln k. This finding suggests that in contrast to horseradish peroxidase, electron transfer is not the rate-limiting step during oxidation by lignin peroxidase compound II. A mechanism is proposed for lignin peroxidase compound II reactions consisting of an equilibrium electron transfer step followed by a subsequent rate-limiting step. Analysis of the correlation coefficients for linear relationships between ln Kd and ln Km and different calculated redox parameters supports a mechanism in which the acidic forms of phenols are oxidized by lignin peroxidase and electron transfer is coupled with proton transfer. 1,2-Dimethoxyarenes did not comply with the trend for phenolic substrates, which may be a result of more than one substrate binding site on lignin peroxidase and/or alternative binding modes. This behavior was supported by analogue studies with the 1,2-dimethoxyarenes veratric acid and veratryl aldehyde, both of which are not oxidized by lignin peroxidase. Inclusion of either had little effect on the rate of oxidation of phenolic substrates yet resulted in a decrease in the oxidation rate of 1,2-dimethoxyarene substrates, which was considerable for veratryl alcohol and less pronounced for 3,4-dimethoxyphenethylalcohol and 3,4-dimethoxycinnamic acid, in particular in the presence of veratric acid.     

Demonstration of Phenolic Compounds in Plant Tissues by An Osmium-Iodide Postfixation Procedure

A simple procedure to stain phenols in plant tissues is described, Postfixation with an aqueous solution prepared by mixing 2 cc of 2% osmium tetroxide and 8 cc of 3% potassium iodide yields brilliant visualization of phenol-containing vacuoles in different tissues of plants (e.g. coffee, oak, tobacco and spruce) bearing high concentration of phenolic compounds. Areas bearing phenols become dark gray to black. Chemical experiments demonstrate that osmium-potassium iodide (Os-KI) mixture reacts rapidly with several naturally occurring plant phenols, developing black solutions from which black solids precipitate. Phenols containing o-dihydroxy groups react with Os-KI solution more rapidly than other structurally different phenols. Therefore, o-dihydroxy units in an aromatic ring seem to function as primary sites of reactivity with the osmium-iodide complexes.     

Meta-pathway degradation of phenolics by thermophilic Bacilli

Thermophilic bacteria capable of degrading phenol as the sole carbon source were isolated from sewage effluent. The isolates were aerobic, sporulating, motile rod-shaped bacteria characterized as Bacillus species with growth temperature optima of 50–60°C. The enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol-2,3-dioxygenase, was detected in all isolates grown in the presence of phenol. One strain, designated Bacillus strain Cro3.2, was capable of degrading phenol, o-, m-, and p-cresol via the meta-pathway and tolerated phenol at concentrations up to 0.1% (w/v) without apparent inhibition of growth. Phenol degradation activities in strain Cro3.2 were induced 3–5 h after supplementation by phenol, orcinol, and the cresols but not by halo- or nitro-substituted phenols. Maximal rates of phenol degradation in stirred bioreactors (10 μmol/min⁻¹/g⁻¹ cells) were achieved at an O₂ delivery rate of 1.0 vvm and temperatures of 45–60°C; however, catechol-2,3-dioxygenase (but not 2-hydroxymuconic semialdehyde dehydrogenase) was rapidly inactivated at high oxygen concentrations. Whole cells of Bacillus strain Cro3.2 entrapped in calcium alginate, polyacrylamide, and agarose gels showed widely different rates of phenol degradation. In calcium alginate gels, rapid loss of phenol-degrading activity was attributed to calcium-induced inactivation of catechol-2,3-dioxygenase. No stabilization with respect to oxygen-induced inactivation was observed under any of the immobilization conditions. It is concluded that the counteractive effects of oxygen limitation at low dO₂ and inactivation of catechol-2,3-dioxygenase at high dO₂ levels pose a significant impediment to the use of resting thermophile cells in the treatment of phenolic waste streams.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1.) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

Numerical and Monte Carlo simulations of phenolic polymerizations catalyzed by peroxidase

Numerical and Monte Carlo simulations of horseradish peroxidase-catalyzed phenolic polymerizations have been performed. Kinetic constants for the simulations were fit to data from the oxidation and polymerization of bisphenol A. Simulations of peroxidase-catalyzed phenolic polymerization were run as a function of enzyme concentration and radical transfer and radical coupling rate constants. Predictions were performed with respect to conversion vs. time and number average molecular weight and polydispersity vs. conversion. It is shown that the enzymatic polymerization of phenols can be optimized with respect to high molecular weights by employing low enzyme concentrations and phenols with low radical coupling rate constants coupled with relatively high radical transfer rate constants. Such phenols may be identified by using linear free energy relationships that relate radical reactivity to electron donating/withdrawing potential of the phenolic substituent. (c) 1993 John Wiley & Sons, Inc.