The chloride-activated peroxidation of catechol as a mechanistic probe of chloroperoxidase reactions. Competitive activation as evidence for a catalytic chloride binding site on compound I

Chloride ion (Cl-) effects on chloroperoxidase (CPO)-catalyzed peroxidation of catechol were used to probe the involvement of Cl- in CPO reactions. High concentrations of Cl- inhibit catechol peroxidation by competing with hydrogen peroxide (KI = 370 mM). However, at lower concentrations, Cl- is a linear competitive activator versus catechol (KDC = 35 mM). Addition of good halogenation substrates to the peroxidatic reaction mixture converts Cl- from a competitive activator to a competitive inhibitor. The KI (10 mM) for this halogenation substrate promoted Cl- inhibition is equivalent to the KM (11 mM) for Cl- in CPO-catalyzed halogenation reactions. During this inhibition, the halogenation substrate is consumed and, at the point where its consumption is complete, Cl- again becomes an activator. Also, at 2.0 mM hydrogen peroxide, CPOs chlorination reaction and its Cl- -activated peroxidatic reaction have similar apparent kcat values. All data are consistent with a mechanism in which Cl- competes with catechol for binding to CPO Compound I. Catechol binding initiates the Cl- -independent path, in which Compound I acts as the oxidizing agent for catechol. When Cl- binds to Compound I, it reacts to yield the enzymatic chlorinating intermediate which is responsible for either the oxidation of catechol in the Cl- -dependent path or the chlorination of substrates in the halogenation pathway. Cl- activation of the peroxidatic reaction is due to a shift from the Cl- -independent pathway to the Cl- -dependent process. The mechanism is unique in that exclusion of the substrate from its primary binding site leads to an increase in the catalytic efficiency of the reaction. This catechol-Cl- system also offers further potential for probing the specificity and chemistry of the key enzymatic intermediates in haloperoxidase-catalyzed reactions.     

Effects of 2-mercaptoacetate in isolated liver mitochondria in vitro. Competitive inhibition of 3-hydroxybutyrate dehydrogenase and depression of the beta-oxidation pathway.

The effects of 2-mercaptoacetate on the respiration rates induced by different substrates were studied in vitro in isolated liver mitochondria. With palmitoyl-L-carnitine or 2-oxoglutarate as the substrate, the ADP-stimulated respiration (State 3) was dose-dependently inhibited by 2-mercaptoacetate. with glutamate or succinate as the substrate. State-3 respiration was only slightly inhibited by 2-mercaptoacetate. In contrast, the oxidation rate of 3-hydroxybutyrate was competitively inhibited by 2-mercaptoacetate in both isolated mitochondria and submitochondrial particles. In uncoupled mitochondria and in mitochondria in which ATP- and GTP-dependent acyl-CoA biosynthesis was inhibited, the inhibitory effect of 2-mercaptoacetate on palmitoyl-L-carnitine oxidation was abolished; under the same conditions, however, inhibition of 3-hydroxybutyrate oxidation by 2-mercaptoacetate still persisted. These results led to the following conclusions: 2-mercaptoacetate itself enters the mitochondrial matrix, inhibits fatty acid oxidation through a mechanism requiring an energy-dependent activation of 2-mercaptoacetate and itself inhibits 3-hydroxybutyrate oxidation through a competitive inhibition of the membrane-bound 3-hydroxybutyrate dehydrogenase. This study also strongly suggests that the compound responsible for the inhibition of fatty acid oxidation is 2-mercaptoacetyl-CoA.     

Reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol dioxygenases

The reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol 1,2-dioxygenase and catechol 2,3-dioxygenase from Pseudomonas putida were examined. Both 3-substituted catechols are oxidized by catechol 2,3-dioxygenase at approximately 30% of the rate observed for catechol oxidation by this enzyme. Analysis of the products of the reactions showed that ring cleavage occurs in a normal fashion between carbons 2 and 3 of the alternate substrates. 3-Ethylcatechol is oxidized by catechol 1,2-dioxygenase at about 6% of the rate of catechol oxidation; ring cleavage occurs between carbons 1 and 2 to give 2-ethyl-cis,cis-muconic acid. However, 3-(methylthio)catechol is a very poor substrate for catechol 1,2-dioxygenase (0.8% of the rate of catechol), but it is a potent competitive inhibitor (Ki = 0.6 microM). The effects of 3-(methylthio)catechol and 3-ethylcatechol on the visible and EPR spectra of catechol 1,2-dioxygenase are also reported.     

Phthalonic acid, an inhibitor of alpha-oxoglutarate transport in mitochondria.

Phthalonic acid is a powerful inhibitor of alpha-oxoglutarate transport in mitochondria. This conclusion is based on the following observations: 1. Phthalonic acid inhibits the oxidation of alpha-oxoglutarate but has no effect on the oxidation of glutamate or cis-aconitate. 2. With arsenite present, phthalonic acid inhibits the oxidation of glutamate plus malate and of cis-aconitate plus malate. Under these conditions alpha-oxoglutarate accumulates inside the mitochondria. With glutamate plus malate as substrates the inhibition is competitive with malate with a Ki value of 20 muM. 3. Phthalonic acid inhibits the oxidation of intramitochondrial NAD(P)H by alpha-oxoglutarate plus ammonia. The inhibition is competitive with respect to alpha-oxoglutarate with a Ki of 30 muM. 4. Phthalonic acid inhibits the exchange between extramitochondrial alpha-oxoglutarate and intramitochondrial malate.     

Regulation of growth of Acinetobacter calcoaceticus NCIB8250 on L-mandelate in batch culture.

Batch culture of Acinetobacter calcoaceticus in L-mandelate- or phenylglyoxylate-salts medium showed an unusual non-exponential pattern unless the inoculum had been grown on benzyl alcohol. There were transient accumulations of benzaldehyde and benzyl alcohol caused by the limitation of L-mandelate oxidation by low activities of benzaldehyde dehydrogenase and the diversion of reducing power to the formation of benzyl alcohol. In vivo enzymic activities were estimated from patterns of substrate utilization in batch cultures containing pairs of substrates. When bacteria previously grown in L-mandelate-salts medium were inoculated into media containing L-mandelate and a second carbon source, metabolism of L-mandelate was arithmetical in the presence of benzoate, catechol or succinate, but accelerated on exhaustion of the second substrate. This indicated repression of the enzymes involved in L-mandelate oxidation. Inoculation of bacteria grown in benzoate-salts medium into medium containing L-mandelate and benzoate gave diauxie with initial utilization of benzoate. Similar experiments showed that benzoate oxidation was not repressed by catechol and only partially repressed by succinate. Measurement of L-mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I in bacterial extracts showed no evidence for feedback inhibition by intermediates of the pathway. The rates of L-mandelate and benzoate utilization by bacterial suspensions were inhibited by succinate and catechol but not by other intermediates of the pathway.     

Internal Binding of Halogenated Phenols in Dehaloperoxidase-Hemoglobin Inhibits Peroxidase Function

Dehaloperoxidase (DHP) from the annelid Amphitrite ornata is a catalytically active hemoglobin-peroxidase that possesses a unique internal binding cavity in the distal pocket above the heme. The previously published crystal structure of DHP shows 4-iodophenol bound internally. This led to the proposal that the internal binding site is the active site for phenol oxidation. However, the native substrate for DHP is 2,4,6-tribromophenol, and all attempts to bind 2,4,6-tribromophenol in the internal site under physiological conditions have failed. Herein, we show that the binding of 4-halophenols in the internal pocket inhibits enzymatic function. Furthermore, we demonstrate that DHP has a unique two-site competitive binding mechanism in which the internal and external binding sites communicate through two conformations of the distal histidine of the enzyme, resulting in nonclassical competitive inhibition. The same distal histidine conformations involved in DHP function regulate oxygen binding and release during transport and storage by hemoglobins and myoglobins. This work provides further support for the hypothesis that DHP possesses an external binding site for substrate oxidation, as is typical for the peroxidase family of enzymes.     

Inhibition of catechol-O-methyltransferase by N-(3,4-dihydroxyphenyl) maleimide

Catechol-O-methyltransferase (COMT) is inhibited rapidly and irreversibly by N-(3,4-dihydroxyphenyl) maleimide. S-adenosylmethionine (AdoMet) and magnesium ions protect the enzyme from inactivation by this compound, but no protection is observed by the catechol substrate. However, the corresponding succinimide analogue shows a reversible inhibition of COMT, which is competitive with pyrocatecholphthalein and non-competitive with AdoMet. Amino-group reagents also inhibit COMT and this inhibition is protected by AdoMet, suggesting that sulphydryl and amino groups essential for activity are located in an AdoMet-binding site on COMT. The maleimide derivative may be considered to be an active-site directed inhibitor.     

Functional interaction of diphenols with polyphenol oxidase. Molecular determinants of substrate/inhibitor specificity

Polyphenol oxidase (PPO) catalyzes the oxidation of o-diphenols to their respective quinones. The quinones autopolymerize to form dark pigments, an undesired effect. PPO is therefore the target for the development of antibrowning and antimelanization agents. A series of phenolic compounds experimentally evaluated for their binding affinity and inhibition constants were computationally docked to the active site of catechol oxidase. Docking studies suggested two distinct modes of binding, dividing the docked ligands into two groups. Remarkably, the first group corresponds to ligands determined to be substrates and the second group corresponds to reversible inhibitors. Analyses of the complexes provide structural explanations for correlating subtle changes in the position and nature of the substitutions on diphenols to their functional properties as substrates and inhibitors. Higher reaction rates and binding are reckoned by additional interactions of the substrates with key residues that line the hydrophobic cavity. The docking results suggest that inhibition of oxidation stems from an interaction between the aromatic carboxylic acid group and the apical His109 of the four coordinates of the trigonal pyramidal coordination polyhedron of CuA. The spatial orientation of the hydroxyl in relation to the carboxylic group either allows a perfect fit in the substrate cavity, leading to inhibition, or because of a steric clash flips the molecule vertically, facilitating oxidation. This is the first study to explain, at the molecular level, the determinants of substrate and inhibitor specificity of a catechol oxidase, thereby providing a platform for the design of selective inhibitors useful to both the food and pharmaceutical industries.     

Inhibition of the catecholase activity of biomimetic dinuclear copper complexes by kojic acid

The inhibition of the catechol oxidase activity exhibited by three dinuclear copper(II) complexes, derived from different diaminotetrabenzimidazole ligands, by kojic acid [5-hydroxy-2-(hydroxymethyl)-γ-pyrone] has been studied. The catalytic mechanism of the catecholase reaction proceeds in two steps and for both of these inhibition by kojic acid is of competitive type. The inhibitor binds strongly to the dicopper(II) complex in the first step and to the dicopper-dioxygen adduct in the second step, preventing in both cases the binding of the catechol substrate. Binding studies of kojic acid to the dinuclear copper(II) complexes and a series of mononuclear analogs, carried out spectrophotometrically and by NMR, enable us to propose that the inhibitor acts as a bridging ligand between the metal centers in the dicopper(II) catalysts. Received: 23 August 1999 / Accepted: 20 January 2000     

Inhibition of intestinal peroxidase activity by nonsteroidal antiinflammatory drugs

The peroxidase activity of the mitochondrial fraction of rat intestine is inhibited in vitro by non-steroidal antiinflammatory drugs (NSAIDs), such as indomethacin (IMN) and acetylsalicylic acid (ASA), the former being more potent than the latter. The peroxidase was solubilised by cetab-NH₄Cl extraction and purified to apparent homogeneity by Sephadex G-150 gel filtration and affinity chromatography on Con-A Sepharose. The purified enzyme activity was 80% inhibited by 150 μM IMN and 50% by 2.67 mM ASA. IMN could also inhibit lactoperoxidase activity to the same extent but not the horseradish peroxidase activity. The inhibition of peroxidase-catalysed iodide oxidation by IMN and ASA was optimal at pH 5.5 and 4.5, respectively. Kinetic studies revealed that the inhibition by IMN was competitive with respect to iodide or guaiacol, while the inhibition by ASA was noncompetitive and reversible in nature. Studies of some structural analogues showed that indole-3-acetic acid was as effective as IMN, while salicylic acid was more potent than ASA. Spectral studies showed a small bathochromic shift of the Soret band of the enzyme by IMN, suggesting its possible interaction at or near the heme moiety. The competitive nature of IMN may be explained as due to its oxidation by the peroxidase to a product absorbing at 412 nm, the formation of which is inhibited by iodide. We suggest that IMN inhibits intestinal peroxidase activity by acting as a competitive substrate for the enzyme. As intestinal peroxidase is mainly contributed by the invading eosinophils, NSAIDs may affect the host defence mechanism by inhibiting the activity of the enzyme.     

A multi-step kinetic model for substrate assimilation and bacterial growth: application to benzene biodegradation

A multi-step kinetic model based on the concept of synthesizing unit (SU) was developed for describing benzene biodegradation in Pseudomonas putida F1. The model herein presented considered substrate arrival rates to the SU rather than concentrations, and provided a reasonable good fit of the dynamics of both catechol and biomass concentrations experimentally determined. It was based on very general assumptions and could be applied to any process accumulating metabolic intermediates. Conventional growth models considering a single step can be regarded as a particular case of this multi-step model. Despite the merits of this model, its applicability strongly depends on the knowledge of the complex induction-repression and inhibition mechanisms governing the different catabolic steps of the degradation pathway, which in most cases are difficult to elucidate experimentally and/or to model mathematically. In this particular case repression of benzene oxidation by catechol and self-inhibition of catechol transformation were experimentally confirmed and considered in the simulation, resulting in a good fit (relative average error of 6%) of the experimental data.     

Inhibition of the oxidation of acetaldehyde and formaldehyde by hepatocytes and mitochondria by crotonaldehyde

Crotonaldehyde was oxidized by disrupted rat liver mitochondrial fractions or by intact mitochondria at rates that were only 10 to 15% that of acetaldehyde. Although a poor substrate for oxidation, crotonaldehyde is an effective inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, by intact mitochondria, and by isolated hepatocytes. Inhibition by crotonaldehyde was competitive with respect to acetaldehyde, and the Ki for crotonaldehyde was about 5 to 20 microM. Crotonaldehyde had no effect on the oxidation of glutamate or succinate. Very low levels of acetaldehyde were detected during the metabolism of ethanol. Crotonaldehyde increased the accumulation of acetaldehyde more than 10-fold, indicating that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, also inhibited the oxidation of acetaldehyde generated by the metabolism of ethanol. Formaldehyde was a substrate for the low-Km mitochondrial aldehyde dehydrogenase, as well as for a cytosolic, glutathione-dependent formaldehyde dehydrogenase. Crotonaldehyde was a potent inhibitor of mitochondrial oxidation of formaldehyde, but had no effect on the activity of formaldehyde dehydrogenase. In hepatocytes, crotonaldehyde produced about 30 to 40% inhibition of formaldehyde oxidation, which was similar to the inhibition produced by cyanamide. This suggested that part of the formaldehyde oxidation occurred via the mitochondrial aldehyde dehydrogenase, and part via formaldehyde dehydrogenase. The fact that inhibition by crotonaldehyde is competitive may be of value since other commonly used inhibitors of aldehyde dehydrogenase are irreversible inhibitors of the enzyme.     

A quick method for the determination of inhibition constants.

The inhibition constant Ki in the common case of competitive inhibition can be obtained by simple comparison of progress curves in the presence and in the absence of inhibitor. The difference between the times taken for the concentration of substrate to fall to the same value is used to obtain Ki. The procedure to use when the product inhibits is described. When there is mixed inhibition, reactions at different substrate concentrations are used to obtain both inhibition constants.     

A two-step model for the kinetics of BTX degradation and intermediate formation by Pseudomonas putida F1

A two-step model is developed for the aerobic biodegradation of benzene,toluene, and p-xylene (BTX) by Pseudomonas putida F1. The model contains three unique features. First, an initial dioxygenation step transforms BTX into their catechol intermediates, but does not support biomassgrowth. Second, the benzene or toluene intermediates are mineralized, which supports biomass synthesis. Third, BTX exhibit competitive inhibition on each other's transformation, while toluene and benzenenoncompetitively inhibit the mineralization of their catechol intermediate. A suite of batch and chemostat experiments is used to systematically measure the kinetic parameters for the two-step transformations and the substrate interactions.     

Calcium ion downregulates soluble guanylyl cyclase activity: evidence for a two-metal ion catalytic mechanism

The inhibition of soluble guanylyl cyclase by Ca2+ has been kinetically characterized and the results support a two-metal-ion catalytic mechanism for formation of cGMP. Ca2+ reversibly inhibits both the basal and NO-stimulated forms of bovine lung soluble guanylyl cyclase. Inhibition is independent of the activator identity and concentration, revealing that Ca2+ interacts with a site independent of the heme regulatory site. Inhibition by Ca2+ is competitive with respect to Mg2+ in excess of substrate, with Kis values of 29 +/- 4 and 6.6 +/- 0.6 microM for the basal and activated states, respectively. Ca2+ inhibits noncompetitively with respect to the substrate MgGTP in both activity states. The qualitatively similar inhibition pattern and quantitatively different Ki values between the basal and NO-stimulated states suggest that the Ca2+ binding site undergoes some structural modification upon activation of the enzyme. The competitive nature of Ca2+ inhibition with respect to excess Mg2+ is consistent with a two-metal-ion mechanism for cyclization.     

Adeno-Associated Virus Type 2 Rep78 Inhibition of PKA and PRKX: Fine Mapping and Analysis of Mechanism

Hormones and neurotransmitters utilize cyclic AMP (cAMP) as a second messenger in signal transduction pathways to regulate cell growth and division, differentiation, gene expression, and metabolism. Adeno-associated virus type 2 (AAV-2) nonstructural protein Rep78 inhibits members of the cAMP signal transduction pathway, the protein kinases PKA and PRKX. We mapped the kinase binding and inhibition domain of Rep78 for PRKX to amino acids (aa) 526 to 561 and that for PKA to aa 526 to 621. These polypeptides were as potent as full-length Rep78 in kinase inhibition, which suggests that the kinase-inhibitory domain is entirely contained in these Rep peptides. Steady-state kinetic analysis of Rep78-mediated inhibition of PKA and PRKX showed that Rep78 appears to increase the K(m) value of the peptide kinase substrate, while the maximal velocity of the reaction was unaffected. This indicates that Rep78 acts as a competitive inhibitor with respect to the peptide kinase substrate. We detected homology between a cellular pseudosubstrate inhibitor of PKA, the protein kinase inhibitor PKI, and the PRKX and PKA inhibition domains of Rep78. Due to this homology and the competitive inhibition mechanism of Rep78, we propose that Rep78 inhibits PKA and PRKX kinase activity by pseudosubstrate inhibition.     

Mechanism of inhibition of dopamine beta-monooxygenase by quinol and phenol derivatives, as determined by solvent and substrate deuterium isotope effects.

The mechanism of interaction of quinols and phenols with dopamine beta-monooxygenase (D beta M) has been investigated. The ratio of quinone formation (from catechol) to oxygen consumption rises from a value of 1 in the presence of phenethylamine substrate to 2 in the absence of substrate. These results implicate quinol oxidation at both the reductant- and substrate-binding sites of D beta M. In the presence of saturating ascorbate, catechol and p-hydroquinol behave as mechanism-based inhibitors of D beta M, with partitioning ratios of turnover to inactivation of 21:1 and 41:1, respectively. Phenol is found to inactivate the enzyme in a manner similar to p-cresol, suggesting that the methyl group of p-cresol is not an essential component of enzyme inhibition. Solvent isotope effects on inactivation and turnover have been measured for various inactivators. Although the majority of these inhibitors, including catechol, p-hydroquinol, aniline, phenethylenediamine, and benzylhydrazine, are characterized by relatively small solvent isotope effects (1.5-2.5) on the inactivation rate constant (ki), solvent isotope effects on ki for phenol and p-cresol are 5.7 and 7.4, respectively. By contrast, solvent isotope effects on the turnover of p-cresol are almost unity. Using p-cresol-d7 as substrate, we observe D(kcat) = 5.2 and D(kcat/Km) = 3.1, while isotope effects on inactivation are D(ki) = 0.95 and D(ki/Ki) = 0.59. These results lead us to propose that inhibitors fall into two mechanistic classes, involving either one-electron oxidation to form radical cation intermediates (quinols) or hydrogen atom abstraction (phenols).(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel mechanism for adenylyl cyclase inhibition from the crystal structure of its complex with catechol estrogen

Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl cyclases and the abundant trans-membrane adenylyl cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl cyclase inhibitors.     

Fluorocitrate inhibition of aconitate hydratase and the tricarboxylate carrier of rat liver mitochondria

1. The effect of biologically synthesized and purified fluorocitrate on the metabolism of tricarboxylate anions by isolated rat liver mitochondria was investigated, in relation to the claim by Eanes et al. (1972) that this fluoro compound inhibits the tricarboxylate carrier at concentrations at which it has little effect on the aconitate hydratase activity. 2. That the inhibitory action of fluorocitrate is at the level of the aconitate hydratase and not at the level of the tricarboxylate carrier is indicated by the following findings. Although the oxidation of citrate and cis-aconitate, but not that of isocitrate, was inhibited by fluorocitrate, the exchange of internal citrate for external citrate or l-malate was not. Had the tricarboxylate carrier been affected, these latter exchange reactions would have been inhibited. 3. By using aconitate hydratase solubilized from mitochondria it was found that with citrate as substrate the inhibition by fluorocitrate was partially competitive (K(i)=3.4x10(-8)m), whereas with cis-aconitate as substrate the inhibition was partially non-competitive (K(i)=3.0x10(-8)m).     

The kinetics of phenethylhydrazine oxidation by monoamine oxidase

1. In the presence of the substrate benzylamine, phenethylhydrazine has been shown to be a competitive inhibitor of monoamine oxidase from rat liver and pig brain. 2. Phenethylhydrazine is also a substrate for monoamine oxidase. Reciprocal plots for hydrazine oxidation give families of intersecting lines in contrast with the parallel lines previously reported for tyramine oxidation. 3. Two possible modifications of the mechanism obeyed by tyramine oxidation are suggested, but the product inhibition results are insufficient to distinguish between these two mechanisms.