Comparative study of catalase-peroxidases (KatGs)

Catalase-peroxidases or KatGs from seven different organisms, including Archaeoglobus fulgidus,Bacillus stearothermophilus, Burkholderia pseudomallei, Escherichia coli, Mycobacterium tuberculosis, Rhodobacter capsulatus and Synechocystis PCC 6803, have been characterized to provide a comparative picture of their respective properties. Collectively, the enzymes exhibit similar turnover rates with the catalase and peroxidase reactions varying between 4900 and 15,900 s⁻¹ and 8-25 s⁻¹, respectively. The seven enzymes also exhibited similar pH optima for the peroxidase (4.25-5.0) and catalase reactions (5.75), and high sensitivity to azide and cyanide with IC₅₀ values of 0.2-20 μM and 50-170 μM, respectively. The K_Ms of the enzymes for H₂O₂ in the catalase reaction were relatively invariant between 3 and 5 mM at pH 7.0, but increased to values ranging from 20 to 225 mM at pH 5, consistent with protonation of the distal histidine (pKa approximately 6.2) interfering with H₂O₂ binding to Cpd I. The catalatic k_cat was 2- to 3-fold higher at pH 5 compared to pH 7, consistent with the uptake of a proton being involved in the reduction of Cpd I. The turnover rates for the INH lyase and isonicotinoyl-NAD synthase reactions, responsible for the activation of isoniazid as an anti-tubercular drug, were also similar across the seven enzymes, but considerably slower, at 0.5 and 0.002 s⁻¹, respectively. Only the NADH oxidase reaction varied more widely between 10⁻⁴ and 10⁻² s⁻¹ with the fastest rate being exhibited by the enzyme from B. pseudomallei.     

Role of Phe113 at the distal side of the heme domain of an oxygen-sensor (Ec DOS) in the characterization of the heme environment

The heme-based oxygen-sensor enzyme from Escherichia coli (Ec DOS) is a heme-regulated phosphodiesterase with activity on cyclic-di-GMP and is composed of an N-terminal heme-bound sensor domain with the PAS structure and a C-terminal functional domain. The activity of Ec DOS is substantially enhanced by the binding of O₂ to the Fe(II)-protoporphyrin IX complex [Fe(II) complex] in the sensor domain. The binding of O₂ to the Fe(II) complex changes the structure of the sensor domain, and this altered structure becomes a signal that is transduced to the functional domain to trigger catalysis. The first step in intra-molecular signal transduction is the binding of O₂ to the Fe(II) complex, and detailed elucidation of this molecular mechanism is thus worthy of exploration. The X-ray crystal structure reveals that Phe113 is located close to the O₂ molecule bound to the Fe(II) complex in the sensor domain. Here, we found that the O₂ association rate constants (>200 × 10⁻³ μM⁻¹ s⁻¹: F113L; 26 × 10⁻³ μM⁻¹ s⁻¹: F113Y) of the Fe(II) complexes of Phe113 mutants were markedly different from that (51 × 10⁻³ μM⁻¹ s⁻¹) of the wild-type enzyme, and auto-oxidation rates (0.00068 min⁻¹: F113L; 0.039 min⁻¹: F113Y) of the Phe113 mutants also differed greatly from that (0.0062 min⁻¹) of the wild-type enzyme. We thus suggest that Phe113, residing near the O₂ molecule, has a critical role in optimizing the Fe(II)-O₂ complex for effective regulation of catalysis by the oxygen-sensor enzyme. Interactions of CO and cyanide anion with the mutant proteins were also studied.     

Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions

GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k_cat/K_M(glucose) = 93 μM⁻¹ s⁻¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k_cat/K_M(glucose) = 27 μM⁻¹ s⁻¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM_ox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FM_ox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k_cat/K_M(FM_ox) = 27 μM⁻¹ s⁻¹ and 17 μM⁻¹ s⁻¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.     

A nanocomposite/crude extract enzyme-based xanthine biosensor

A novel amperometric biosensor for xanthine was developed based on covalent immobilization of crude xanthine oxidase (XOD) extracted from bovine milk onto a hybrid nanocomposite film via glutaraldehyde. Toward the preparation of the film, a stable colloids solution of core–shell Fe₃O₄/polyaniline nanoparticles (PANI/Fe₃O₄ NPs) was dispersed in solution containing chitosan (CHT) and H₂PtCl₆ and electrodeposited over the surface of a carbon paste electrode (CPE) in one step. Scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectrophotometry, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used for characterization of the electrode surface. The developed biosensor (XOD/CHT/Pt NPs/PANI/Fe₃O₄/CPE) was employed for determination of xanthine based on amperometric detection of hydrogen peroxide (H₂O₂) reduction at –0.35 V (vs. Ag/AgCl). The biosensor exhibited a fast response time to xanthine within 8 s and a linear working concentration range from 0.2 to 36.0 μM (R² = 0.997) with a detection limit of 0.1 μM (signal/noise [S/N] = 3). The sensitivity of the biosensor was 13.58 μA μM⁻¹ cm⁻². The apparent Michaelis–Menten (K_m) value for xanthine was found to be 4.7 μM. The fabricated biosensor was successfully applied for measurement of fish and chicken meat freshness, which was in agreement with the standard method at the 95% confidence level.     

A synthetic dinuclear copper(II) hydrolase and its potential as antitumoral: Cytotoxicity, cellular uptake, and DNA cleavage

We have studied the protonation equilibria of a dicopper(II) complex [Cu₂(μ-OH)(C₂₁H₃₃ON₆)](ClO₄)₂·H₂O, (1), in aqueous solution, its interactions with DNA, its cytotoxic activity, and its uptake in tumoral cells. C₂₁H₃₃ON₆ corresponds to the ligand 4-methyl-2,6-bis[(6-methyl-1,4-diazepan-6-yl)iminomethyl]phenol. From spectrophotometric data the following pKa values were calculated 3.27, 4.80 and 6.10. Complex 1 effectively promotes the hydrolytic cleavage of double-strand plasmid DNA under anaerobic and aerobic conditions. The following kinetic parameters were calculated k_cat of 2.73 × 10⁻⁴ s⁻¹, K_M of 1.36 × 10⁻⁴ M and catalytic efficiency of 2.01 s⁻¹ M⁻¹, a 2.73 × 10⁷ fold increase in the rate of the reaction compared to the uncatalyzed hydrolysis rate of DNA. Competition assays with distamycin reveal minor groove binding. Complex 1 inhibited the growth of two tumoral cell lines, GLC4 and K562, with the IC₅₀ values of 14.83 μM and 34.21 μM, respectively. There is a good correlation between cell growth inhibition and intracellular copper content. When treated with 1, cells accumulate approximately twice as much copper as with CuCl₂. Copper-DNA adducts are formed inside cells when they are exposed to the complex. In addition, at concentrations that compound 1 inhibits tumoral cell growth it does not affect macrophage viability. These results show that complex 1 has a good therapeutic prospect.     

Nitrilotris(methylenephosphonates) in aqueous solution and solid state - dilatometric, potentiometric and NMR investigations

Dissociation and alkali complex formation equilibria of nitrilotris(methylenephosphonic acid) (NTMP, H₆L) have been studied by dilatometric, potentiometric and ³¹P NMR-controlled titrations. Dilatometry indicated the formation of alkali complexes ML (M=Li, Na, K, Rb, Cs) at high pH with a stability decreasing from Li to Cs. An efficient combination of potentiometric and NMR methods confirmed two types of alkali metal complexes MHL and ML. Stability constants for the equilibria following M⁺ + HL⁵⁻ ? MHL⁴⁻ and M⁺ + L⁶⁻ ? ML⁵⁻, respectively, were determined: logK_NaHL=1.08(0.07), logK_KHL=0.86(0.08), logK_NaL=2.24(0.03). Systematic errors are introduced by using alkali metal hydroxides as titrants for routine potentiometric determinations of dissociation constants pK_a5^app and pK_a6^app. Correction formulae were derived to convert actual dissociation constants pK_a into apparent dissociation constants pK_a^app (or vice versa). The actual dissociation constants were found: pK_a5(H₂L⁴⁻ ? H⁺ + HL⁵⁻)=7.47(0.03) and pK_a6(HL⁵⁻ ? H⁺ + L⁶⁻)=14.1(0.1). The anisotropy of ³¹P chemical shifts of salts M_nH_6 − nL (M=Li, Na, n=0-5) is more sensitive towards titration (n) than isotropic solution state chemical shifts.     

Structural and equilibrium studies on mixed ligand complexes of Co(II), Ni(II), Cu(II) and Zn(II) with N-(2-benzimidazolyl)methyliminodiacetic acid and typical N, N donor ligands

Mixed ligand complexes: [Co(L)(bipy)] · 3H₂O (1), [Ni(L)(phen)] · H₂O (2), [Cu(L)(phen)] · 3H₂O (3) and [Zn(L)(bipy)] · 3H₂O (4), where L²⁻ = two -COOH deprotonated dianion of N-(2-benzimidazolyl)methyliminodiacetic acid (H₂bzimida, hereafter, H₂L), bipy = 2,2′ bipyridine and phen = 1,10-phenanthroline have been isolated and characterized by elemental analysis, spectral and magnetic measurements and thermal studies. Single crystal X-ray diffraction studies show octahedral geometry for 1, 2 and 4 and square pyramidal geometry for 3. Equilibrium studies in aqueous solution (ionic strength I = 10⁻¹ mol dm⁻³ (NaNO₃), at 25 ± 1 °C) using different molar proportions of M(II):H₂L:B, where M = Co, Ni, Cu and Zn and B = phen, bipy and en (ethylene diamine), however, provides evidence of formation of mononuclear and binuclear binary and mixed ligand complexes: M(L), M(H₋₁L)⁻, M(B)²⁺, M(L)(B), M(H₋₁L)(B)⁻, M₂(H₋₁L)(OH), (B)M(H₋₁L)M(B)⁺, where H₋₁L³⁻ represents two -COOH and the benzimidazole N₁-H deprotonated quadridentate (O⁻, N, O⁻, N), or, quinquedentate (O⁻, N, O⁻, N, N⁻) function of the coordinated ligand H₂L. Binuclear mixed ligand complex formation equilibria: M(L)(B) + M(B)²⁺ ? (B)M(H₋₁L)M(B)⁺ + H⁺ is favoured with higher π-acidity of the B ligands. For Co(II), Ni(II) and Cu(II), these equilibria are accompanied by blue shift of the electronic absorption maxima of M(II) ions, as a negatively charged bridging benzimidazolate moiety provides stronger ligand field than a neutral one. Solution stability of the mixed ligand complexes are in the expected order: Co(II) < Ni(II) < Cu(II) > Zn(II). The Δ log K_M values are less negetive than their statistical values, indicating favoured formation of the mixed ligand complexes over the binary ones.     

The base exchange reaction of NAD+ glycohydrolase: identification of novel heterocyclic alternative substrates

ADP-ribosyl cyclase and NAD⁺ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD⁺, nicotinamide adenine dinucleotide phosphate (NADP⁺) or nicotinamide mononucleotide (NMN⁺) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (k_cat/K_M = 0.9-10 μM⁻¹ s⁻¹) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the k_cat/K_M values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM⁻¹ s⁻¹). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN⁺, k_cat/K_M = 0.05-0.4 μM⁻¹ s⁻¹. The k_cat/K_M values for the exchange reaction with NMN⁺ were generally similar (0.04-0.12 μM⁻¹ s⁻¹) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The k_cat/K_M values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.     

The kinetics of the complex formation between iron(III)-ethylenediaminetetraacetate and hydrogen peroxide in aqueous solution

The kinetics of the formation of the purple complex [Fe^III(EDTA)O₂]³⁻, between Fe^III-EDTA and hydrogen peroxide was studied as a function of pH (8.22-11.44) and temperature (10-40 °C) in aqueous solutions using a stopped-flow method. The reaction was first-order with respect to both reactants. The observed second-order rate constants decrease with an increase in pH and appear to be related to deprotonation of Fe^III-EDTA ([Fe(EDTA)H₂O]⁻ ⇔ Fe(EDTA)OH]²⁻ + H⁺). The rate law for the formation of the complex was found to be d[Fe^IIIEDTAO₂]³⁻/dt=[(k₄[H⁺]/([H⁺] + K₁)][Fe^III-EDTA][H₂O₂], where k₄=8.15±0.05×10⁴ M⁻¹ s⁻¹ and pK₁=7.3. The steps involved in the formation of [Fe(EDTA)O₂]³⁻ are briefly discussed.     

Oxidation of thiocyanate by iron(V) in alkaline medium

The oxidation of thiocyanate by iron(V) (Fe(V)) was studied as a function of pH in alkaline solutions by a premix pulse radiolysis technique. The rates decrease with an increase in pH. The rate law for the oxidation of SCN⁻ by Fe(V) was obtained as −d[Fe(V)]/dt = k₁₀{[H⁺]²/([H⁺]² + K₂[H⁺] + K₂K₃)}[Fe(V)][SCN⁻], where k₁₀ = 5.72 ± 0.19 × 10⁶ M⁻¹ s⁻¹, pK₂ = 7.2, and pK₃ = 10.1. The reaction precedes via a two-electron oxidation, which converts Fe(V) to Fe(III). Thiocyanate reacts approximately 10³× faster with iron(V) than does with iron(VI).     

Peroxynitrite detoxification by ferryl Mycobacterium leprae truncated hemoglobin O

During infection, Mycobacterium leprae is faced with the host macrophagic environment limiting the growth of the bacilli. However, (pseudo-)enzymatic detoxification systems, including truncated hemoglobin O (Ml-trHbO), could allow this mycobacterium to persist in vivo. Here, kinetics of peroxynitrite (ONOOH/ONOO⁻) detoxification by ferryl Ml-trHbO (Ml-trHbOFe(IV)O), obtained by treatment with H₂O₂, is reported. Values of the second-order rate constant for peroxynitrite detoxification by Ml-trHbOFe(IV)O (i.e., of Ml-trHbOFe(III) formation; k_on), at pH 7.2 and 22.0 °C, are 1.5 × 10⁴ M⁻¹ s⁻¹, and 2.2 × 10⁴ M⁻¹ s⁻¹, in the absence of and presence of physiological levels of CO₂ (∼1.2 × 10⁻³ M), respectively. Values of k_on increase on decreasing pH with a pK_a value of 6.7, this suggests that ONOOH reacts preferentially with Ml-trHbOFe(IV)O. In turn, peroxynitrite acts as an antioxidant of Ml-trHbOFe(IV)O, which could be responsible for the oxidative damage of the mycobacterium. As a whole, Ml-trHbO can undertake within the same cycle H₂O₂ and peroxynitrite detoxification.     

Chemiluminescent determination of H2O2 using 4-(1,2,4-triazol-1-yl)phenol as an enhancer based on the immobilization of horseradish peroxidase onto magnetic beads

This article describes the employment of a novel p-phenol derivative, 4-(1,2,4-triazol-1-yl)phenol (TRP), as a highly potent signal enhancer of the luminol-hydrogen peroxide (H₂O₂)-horseradish peroxidase (HRP) chemiluminescence (CL) system. The CL reaction conditions were optimized, and the enhancement characteristics of TRP were compared with those of p-iodophenol (PIP). TRP produced a strong enhancement of the CL with the effect of prolonging the light emission. The developed system was then applied to the determination of H₂O₂ with immobilized HRP using magnetic beads as a solid support. The linear range for H₂O₂ was 2.0 × 10⁻⁶ to 1.0 × 10⁻³ M. The detection limit for H₂O₂ was 2.0 × 10⁻⁶ M. The proposed sensor was applied successfully to the determination of H₂O₂ in rainwater.     

Equilibrium and structural studies on Co(II), Ni(II), Cu(II) and Zn(II) complexes with N-(2-benzimidazolyl)methyliminodiacetic acid: crystal structures of Ni(II) and Cu(II) complexes

Combined pH-metric, UV-Vis, ¹H NMR and EPR spectral investigations on the complex formation of M(II) ions (M=Co, Ni, Cu and Zn) with N-(2-benzimidazolyl)methyliminodiacetic acid (H₂bzimida, hereafter H₂L) in aqueous solution at a fixed ionic strength, I=10⁻¹ mol dm⁻³, at 25 ± 1 °C indicate the formation of M(L), M(H₋₁L)⁻ and M₂(H₋₁L)⁺ complexes. Proton-ligand and metal-ligand constants and the complex formation equilibria have been elucidated. Solid complexes, [M(L)(H₂O)₂] · nH₂O (n=1 for M = Co and Zn, n=2 for M = Ni) and {Cu (μ-L) · 4H₂O}_n, have been isolated and characterized by elemental analysis, spectral, conductance and magnetic measurements and thermal studies. Structures of [Ni(L)(H₂O)₂] · 2H₂O and {Cu(μ-L) · 4H₂O}_n have been determined by single crystal X-ray diffraction. The nickel(II) complex exists in a distorted octahedral environment in which the metal ion is coordinated by the two carboxylate O atoms, the amino-N atom of the iminodiacetate moiety and the pyridine type N-atom of the benzimidazole moiety. Two aqua O atoms function as fifth and sixth donor atoms. The copper(II) complex is made up of interpenetrating polymeric chains of antiferromagnetically coupled Cu(II) ions linked by carboxylato bridges in syn-anti (apical-equatorial) bonding mode and stabilized via interchain hydrogen bonds and π-π stacking interactions.     

Kinetic studies of the reaction of heme-thiolate enzyme chloroperoxidase with peroxynitrite

The kinetics of the reaction of chloroperoxidase with peroxynitrite was studied under neutral and acidic pH by stopped-flow spectrophotometry. Chloroperoxidase catalyzed peroxynitrite decay with the rate constant, k_c, increasing with decreasing pH. The values of k_c obtained at pH 5.1, 6.1 and 7.1 were equal to: (1.96 ± 0.03) × 10⁶, (1.63 ± 0.04) × 10⁶ and (0.71 ± 0.01) × 10⁶ M⁻¹ s⁻¹, respectively. Chloroperoxidase was converted to compound II by peroxynitrite with pH-dependent rate constants: (12.3 ± 0.4) × 10⁶ and (3.8 ± 0.3) × 10⁶ M⁻¹ s⁻¹ at pH 5.1 and 7.1, respectively. After most of peroxynitrite had disappeared, the conversion of compound II into the ferric form of chloroperoxidase was observed. The recovery of the native enzyme was completed within 1 s and 5 s at pH 5.1 and 7.1, respectively. The possible reaction mechanisms of the catalytic decomposition of peroxynitrite by chloroperoxidase are discussed.     

A kinetic investigation of the oxidative addition reactions of the dimeric Bu4N[Ir2(μ-Dcbp)(cod)2] complex with iodomethane

The kinetic results of the oxidative addition of iodomethane to Bu₄N[Ir₂(μ-Dcbp)(cod)₂] (Dcbp = 3,5-dicarboxylatepyrazolate anion) show that oxidative addition can occur via a direct equilibrium pathway (K₁ = 88(22) acetone, 51(3) 1,2-dichloroethane, 55(4) dichloromethane, 52(12) acetonitrile and 43(5) M⁻¹ chloroform) or a solvent-assisted pathway (k₂, k₃). Oxidative addition occurs mainly along the direct pathway, which is a factor 10-40 faster than the solvent-assisted pathway. The observed solvent effect cannot be attributed to the donosity or polarity of the solvents. The fairly negative ΔS^≠ value (−110(7) J K⁻¹ mol⁻¹) and the positive ΔH^≠ value (+47(2) kJ mol⁻¹) for the oxidative addition step are indicative of an associative process.     

Steady-state kinetic mechanism of LodA,a novel cysteine tryptophylquinone-dependent oxidase

LodA is a novel lysine-ε-oxidase which possesses a cysteine tryptophylquinone cofactor. It is the first tryptophylquinone enzyme known to function as an oxidase. A steady-state kinetic analysis shows that LodA obeys a ping-pong kinetic mechanism with values of k_cat of 0.22 ± 0.04 s⁻¹, K_lysine of 3.2 ± 0.5 μM and K_O2 of 37.2 ± 6.1 μM. The k_cat exhibited a pH optimum at 7.5 while k_cat/K_lysine peaked at 7.0 and remained constant to pH 8.5. Alternative electron acceptors could not effectively substitute for O₂ in the reaction. A mechanism for the reductive half reaction of LodA is proposed that is consistent with the ping-pong kinetics.     

Characterization of recombinant UDP-galactopyranose mutase from Aspergillus fumigatus

UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose, which is an important cell wall component in Aspergillus fumigatus and other pathogenic microbes. A. fumigatus UGM (AfUGM) was expressed in Escherichia coli and purified to homogeneity. The enzyme was shown to function as a homotetramer by size-exclusion chromatography and to contain ∼50% of the flavin in the active reduced form. A k_cat value of 72 ± 4 s⁻¹ and a K_M value of 110 ± 15 μM were determined with UDP-galactofuranose as substrate. In the oxidized state, AfUGM does not bind UDP-galactopyranose, while UDP and UDP-glucose bind with K_d values of 33 ± 9 μM and 90 ± 30 μM, respectively. Functional and structural differences between the bacterial and eukaryotic UGMs are discussed.     

Large catalase based bioelectrode for biosensor application

A large catalase (CAT) (Mr ~ 90 kDa), immobilized on multiwalled carbon nanotubes—Nafion^® (MWCNT-NF) matrix and encapsulated with polyethylenimine (PEI) on glassy carbon electrode (GCE), showed a pair of nearly reversible cyclic voltammetric peaks for Fe^(III)/Fe^(II) couple with formal potential of about −0.45 V (vs. Ag/AgCl electrode at pH 7.5). PEI significantly reduced the charge transfer resistance and stabilized the bioelectrode through electrostatic interaction. The electron transfer rate constant and surface coverage of the immobilized CAT were 1.05 ± 0.2 s⁻¹ and 2.1 × 10⁻¹⁰ mol cm⁻², respectively. Studies on electrocatalytic activity and kinetics of GCE/MWCNT-NF/CAT/PEI for hydrogen peroxide (H₂O₂) showed the apparent Michaelis-Menten constant of 3 mM, linear response in the range of 10 μM to 5 mM, response time of ~ 2 s for steady state current, and detection limit of ~ 1 μM. A high operational and storage stability was also demonstrated for the bioelectrode. Hence, the direct electrochemistry of the large catalase and its potential biosensor application have been established through this investigation.     

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.