Rate-limiting Step Of Reaction Research Articles

Heterogeneous Mercury Oxidation on Au(111) from First Principles

Density functional theory (DFT) studies of mercury oxidation on Au(111) are conducted to determine the potential Hg oxidation mechanisms taking place on catalytic gold surfaces by using the Perdew and Wang approximation (PW91) described by a generalized gradient approximation (GGA). The Hg oxidation was examined via a Langmuir-Hinshelwood mechanism where each Hg(0) and Cl2 (or HCl) species is separately adsorbed on the gold surface and the bimolecular reaction occurs through the formation of bound HgCl and HgCl2. For this, the Climbing Image-Nudged Elastic Band (CI-NEB) method has been employed to calculate the activation energies of HgCl and HgCl2 formation pathways. In the three-step Hg oxidation mechanism (Hg → HgCl → HgCl2), the second Cl attachment step is endothermic which is the reaction rate-limiting step, while the first Cl attachment step is exothermic. This observation implies that Hg oxidation prefers a pathway in which HgCl and HgCl2 are formed, rather than a pathway directly oxidizing Hg to HgCl2. In the presence of H atoms due to HCl dissociation on the Au surface, the H atoms lower the activation energy for Hg oxidation by consuming the electron charge of Au atoms, thereby weakening the strength of interaction between Cl and the Au surface and lowering an energy required to detach Cl from the Au surface. This mechanism is in the absence of site competition on the Au surface. In addition, details of the electronic properties of these systems are discussed.

Enhanced Chlorine Dioxide Decay in the Presence of Metal Oxides: Relevance to Drinking Water Distribution Systems

Chlorine dioxide (ClO2) decay in the presence of typical metal oxides occurring in distribution systems was investigated. Metal oxides generally enhanced ClO2 decay in a second-order process via three pathways: (1) catalytic disproportionation with equimolar formation of chlorite and chlorate, (2) reaction to chlorite and oxygen, and (3) oxidation of a metal in a reduced form (e.g., cuprous oxide) to a higher oxidation state. Cupric oxide (CuO) and nickel oxide (NiO) showed significantly stronger abilities than goethite (α-FeOOH) to catalyze the ClO2 disproportionation (pathway 1), which predominated at higher initial ClO2 concentrations (56-81 μM). At lower initial ClO2 concentrations (13-31 μM), pathway 2 also contributed. The CuO-enhanced ClO2 decay is a base-assisted reaction with a third-order rate constant of 1.5 × 10(6) M(-2) s(-1) in the presence of 0.1 g L(-1) CuO at 21 ± 1 °C, which is 4-5 orders of magnitude higher than in the absence of CuO. The presence of natural organic matter (NOM) significantly enhanced the formation of chlorite and decreased the ClO2 disproportionation in the CuO-ClO2 system, probably because of a higher reactivity of CuO-activated ClO2 with NOM. Furthermore, a kinetic model was developed to simulate CuO-enhanced ClO2 decay at various pH values. Model simulations that agree well with the experimental data include a pre-equilibrium step with the rapid formation of a complex, namely, CuO-activated Cl2O4. The reaction of this complex with OH(-) is the rate-limiting and pH-dependent step for the overall reaction, producing chlorite and an intermediate that further forms chlorate and oxygen in parallel. These novel findings suggest that the possible ClO2 loss and the formation of chlorite/chlorate should be carefully considered in drinking water distribution systems containing copper pipes.

Preparation and electrochemical characterization of Ruddlesden–Popper oxide La4Ni3O10 cathode for IT-SOFCs by sol–gel method

La4Ni3O10 oxide was synthesized as a cathode material for intermediate-temperature solid oxide fuel cells by a facile sol–gel method using a nonionic surfactant (EO)106(PO)70(EO)106 tri-block copolymer (F127) as the chelating agent. The crystal structure, electrical conductivity, and electrochemical properties of La4Ni3O10 were investigated by X-ray diffraction, DC four-probe method, electrochemical impedance spectra, and I–V measurements. The La4Ni3O10 cathode showed a significantly low polarization resistance (0.26 Ω cm2) and cathodic overpotential value (0.037 V at the current density of 0.1 A cm−2) at 750 °C. The results measured suggest that the diffusion process was the rate-limiting step for the oxygen reduction reaction. The La4Ni3O10 cathode revealed a high exchange current density value of 62.4 mA cm−2 at 750 °C. Furthermore, an anode-supported single cell with La4Ni3O10 cathode was fabricated and tested from 650 to 800 °C with humidified hydrogen (∼3 vol% H2O) as the fuel and the static air as the oxidant. The maximum power density of 900 mW cm−2 was achieved at 750 °C.

Problems of the stereoselectivity in the norbornadiene [2+2]-cyclodimerization reactions catalyzed by hydride nickel(i) complexes. Theoretical aspects

Cyclodimerization of norbornadiene (NBD) yielding pentacyclic products of exo-trans(cis)endo-structure in the presence of the model catalytically active complex Ni(H)(η4-NBD) has been studied using the DFT/PBE method. The rate-limiting reaction step is the reductive elimination of the metallacycle, the decomposition of the latter yields the norbornadiene dimer.

Design, synthesis and in vitro kinetic study of tranexamic acid prodrugs for the treatment of bleeding conditions

Based on density functional theory (DFT) calculations for the acid-catalyzed hydrolysis of several maleamic acid amide derivatives four tranexamic acid prodrugs were designed. The DFT results on the acid catalyzed hydrolysis revealed that the reaction rate-limiting step is determined on the nature of the amine leaving group. When the amine leaving group was a primary amine or tranexamic acid moiety, the tetrahedral intermediate collapse was the rate-limiting step, whereas in the cases by which the amine leaving group was aciclovir or cefuroxime the rate-limiting step was the tetrahedral intermediate formation. The linear correlation between the calculated DFT and experimental rates for N-methylmaleamic acids 1-7 provided a credible basis for designing tranexamic acid prodrugs that have the potential to release the parent drug in a sustained release fashion. For example, based on the calculated B3LYP/6-31G(d,p) rates the predicted t1/2 (a time needed for 50% of the prodrug to be converted into drug) values for tranexamic acid prodrugs ProD 1-ProD 4 at pH 2 were 556h [50.5h as calculated by B3LYP/311+G(d,p)] and 6.2h as calculated by GGA: MPW1K), 253h, 70s and 1.7h, respectively. Kinetic study on the interconversion of the newly synthesized tranexamic acid prodrug ProD 1 revealed that the t1/2 for its conversion to the parent drug was largely affected by the pH of the medium. The experimental t1/2 values in 1N HCl, buffer pH 2 and buffer pH 5 were 54min, 23.9 and 270h, respectively.

The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle

MnmE is a homodimeric multi-domain GTPase involved in tRNA modification. This protein differs from Ras-like GTPases in its low affinity for guanine nucleotides and mechanism of activation, which occurs by a cis, nucleotide- and potassium-dependent dimerization of its G-domains. Moreover, MnmE requires GTP hydrolysis to be functionally active. However, how GTP hydrolysis drives tRNA modification and how the MnmE GTPase cycle is regulated remains unresolved. Here, the kinetics of the MnmE GTPase cycle was studied under single-turnover conditions using stopped- and quench-flow techniques. We found that the G-domain dissociation is the rate-limiting step of the overall reaction. Mutational analysis and fast kinetics assays revealed that GTP hydrolysis, G-domain dissociation and Pi release can be uncoupled and that G-domain dissociation is directly responsible for the ‘ON’ state of MnmE. Thus, MnmE provides a new paradigm of how the ON/OFF cycling of GTPases may regulate a cellular process. We also demonstrate that the MnmE GTPase cycle is negatively controlled by the reaction products GDP and Pi. This feedback mechanism may prevent inefficacious GTP hydrolysis in vivo. We propose a biological model whereby a conformational change triggered by tRNA binding is required to remove product inhibition and initiate a new GTPase/tRNA-modification cycle.

SMET: Systematic multiple enzyme targeting – a method to rationally design optimal strains for target chemical overproduction

Identifying multiple enzyme targets for metabolic engineering is very critical for redirecting cellular metabolism to achieve desirable phenotypes, e.g., overproduction of a target chemical. The challenge is to determine which enzymes and how much of these enzymes should be manipulated by adding, deleting, under-, and/or over-expressing associated genes. In this study, we report the development of a systematic multiple enzyme targeting method (SMET), to rationally design optimal strains for target chemical overproduction. The SMET method combines both elementary mode analysis and ensemble metabolic modeling to derive SMET metrics including l-values and c-values that can identify rate-limiting reaction steps and suggest which enzymes and how much of these enzymes to manipulate to enhance product yields, titers, and productivities. We illustrated, tested, and validated the SMET method by analyzing two networks, a simple network for concept demonstration and an Escherichia coli metabolic network for aromatic amino acid overproduction. The SMET method could systematically predict simultaneous multiple enzyme targets and their optimized expression levels, consistent with experimental data from the literature, without performing an iterative sequence of single-enzyme perturbation. The SMET method was much more efficient and effective than single-enzyme perturbation in terms of computation time and finding improved solutions.

Oxamate Is an Alternative Substrate for Pyruvate Carboxylase from Rhizobium etli

Oxamate, an isosteric and isoelectronic inhibitory analogue of pyruvate, enhances the rate of enzymatic decarboxylation of oxaloacetate in the carboxyl transferase domain of pyruvate carboxylase (PC). It is unclear, though, how oxamate exerts a stimulatory effect on the enzymatic reaction. Herein, we report direct (13)C nuclear magnetic resonance (NMR) evidence that oxamate acts as a carboxyl acceptor, forming a carbamylated oxamate product and thereby accelerating the enzymatic decarboxylation reaction. (13)C NMR was used to monitor the PC-catalyzed formation of [4-(13)C]oxaloacetate and subsequent transfer of (13)CO(2) from oxaloacetate to oxamate. In the presence of oxamate, the apparent K(m) for oxaloacetate is artificially suppressed (from 15 to 4-5 μM). Interestingly, the steady-state kinetic analysis of the initial rates determined at varying concentrations of oxaloacetate and fixed concentrations of oxamate revealed initial velocity patterns inconsistent with a simple ping-pong-type mechanism. Rather, the patterns suggest the existence of an alternate decarboxylation pathway in which an unstable intermediate is formed. The steady-state kinetic analysis coupled with the normal (13)(V/K) kinetic isotope effect observed on C-4 of oxaloacetate [(13)(V/K) = 1.0117 ± 0.0005] indicates that the transfer of CO(2) from carboxybiotin to oxamate is the partially rate-limiting step of the enzymatic reaction. The catalytic mechanism proposed for the carboxylation of oxamate is similar to that proposed for the carboxylation of pyruvate, which occurs via the formation of an enol intermediate.

Insight into Dimethyl Ether Carbonylation Reaction over Mordenite Zeolite from in-Situ Solid-State NMR Spectroscopy

Carbonylation of dimethyl ether (DME) with CO over H-mordenite (H-MOR) zeolites from 423 to 573 K was studied by in-situ C-13 solid-state NMR spectroscopy. The reaction was monitored separately in the 8-membered ring (MR) and in the 12-MR channels of the zeolite under identical conditions. The experimental results indicated that both 8-MR and 12-MR channels were capable of producing methyl acetate product but with remarkably different selectivities. At low-reaction temperature, surface acetyl species (CH3CO-) as stabilized acylium cation was solely identified in the 8-MR channels, which was confirmed by measurement of its characteristic chemical shift anisotropy (CSA) parameters. Additionally, the intermediate role of the acetyl species was evidenced by the fact that it could react with DME to form methyl acetate. This demonstrated the theoretically proposed specificity of the 8-MR channels for generation and stabilization of the acetyl intermediate which was considered as the rate-limiting step of carbonylation reaction. While in the 12-MR channels, the acetyl intermediate was not found during the reaction, and formation of hydrocarbons was favored. The absence of acetyl intermediate might account for the lower reactivity of the 12-MR channels to generate methyl acetate product.

Transient kinetic studies reveal isomerization steps along the kinetic pathway of Thermus thermophilus 3‐isopropylmalate dehydrogenase

To identify the rate-limiting step(s) of the 3-isopropylmalate dehydrogenase-catalysed reaction, time courses of NADH production were followed by stopped flow (SF) and quenched flow (QF). The steady state kcat and Km values did not vary between enzyme concentrations of 0.1 and 20 μm. A burst phase of NADH formation was shown by QF, indicating that the rate-limiting step occurs after the redox step. The kinetics of protein conformational change(s) induced by the complex of 3-isopropylmalate with Mg(2+) were followed by using the fluorescence resonance energy transfer signal between protein tryptophan(s) and the bound NADH. A reaction scheme was proposed by incorporating the rate constant of a fast protein conformational change (possibly domain closure) derived from the separately recorded time-dependent formation of the fluorescence resonance energy transfer signal. The rate-limiting step seems to be another slower conformational change (domain opening) that allows product release.

The Interaction of the Chemotherapeutic Drug Chlorambucil with Human Glutathione Transferase A1-1: Kinetic and Structural Analysis

Glutathione transferases (GSTs) are enzymes that contribute to cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs. In the present work we investigated the interaction of the chemotherapeutic drug chlorambucil (CBL) with human GSTA1-1 (hGSTA1-1) using kinetic analysis, protein crystallography and molecular dynamics. In the presence of GSH, CBL behaves as an efficient substrate for hGSTA1-1. The rate-limiting step of the catalytic reaction between CBL and GSH is viscosity-dependent and kinetic data suggest that product release is rate-limiting. The crystal structure of the hGSTA1-1/CBL-GSH complex was solved at 2.1 Å resolution by molecular replacement. CBL is bound at the H-site attached to the thiol group of GSH, is partially ordered and exposed to the solvent, making specific interactions with the enzyme. Molecular dynamics simulations based on the crystal structure indicated high mobility of the CBL moiety and stabilization of the C-terminal helix due to the presence of the adduct. In the absence of GSH, CBL is shown to be an alkylating irreversible inhibitor for hGSTA1-1. Inactivation of the enzyme by CBL followed a biphasic pseudo-first-order saturation kinetics with approximately 1 mol of CBL per mol of dimeric enzyme being incorporated. Structural analysis suggested that the modifying residue is Cys112 which is located at the entrance of the H-site. The results are indicative of a structural communication between the subunits on the basis of mutually exclusive modification of Cys112, indicating that the two enzyme active sites are presumably coordinated.

Prodrugs for masking bitter taste of antibacterial drugs—a computational approach

DFT calculations for the acid-catalyzed hydrolysis of several maleamic acid amide derivatives revealed that the reaction rate-limiting step is determined on the nature of the amine leaving group. Further, it was established that when the amine leaving group was a secondary amine, acyclovir or cefuroxime moiety the tetrahedral intermediate formation was the rate-limiting step such as in the cases of acyclovir ProD 1- ProD 4 and cefuroxime ProD 1- ProD 4. In addition, the linear correlation between the calculated and experimental rates provided a credible basis for designing prodrugs for masking bitter taste of the corresponding parental drugs which have the potential to release the parent drug in a sustained release fashion. For example, based on the DFT calculated rates the predicted t₁/₂ (a time needed for 50 % of the reactant to be hydrolyzed to products) for cefuroxime prodrugs, cefuroxime ProD 1- ProD 4, were 12 min, 18 min, 200 min and 123 min, respectively.

Hydrogen oxidation at the Pt–BaZr0.1Ce0.7Y0.1Yb0.1O3−δ (BZCYYb) interface

An asymmetric cell based on a proton conductor, BaZr(0.1)Ce(0.7)Y(0.1)Yb(0.1)O(3-δ) (BZCYYb), with a well-defined patterned Pt electrode was prepared to study the kinetics and mechanism of the hydrogen oxidation reaction under typical conditions for fuel cell operation and hydrogen separation, including operating temperature and hydrogen partial pressure. Steady-state polarization curves were carefully analyzed to determine the apparent exchange current density, limiting current density, and charge transfer coefficients. The empirical reaction order, as estimated from the dependence of electrode polarization (R(p)) and exchange current density on the partial pressure of hydrogen (P(H(2))), varied from 0.55 to 0.71. The results indicate that hydrogen dissociation contributes the most to the rate-limiting step of the hydrogen oxidation reaction taking place at the Pt-BZCYYb interface. At high current densities, surface diffusion of electroactive species appears to contribute to the rate-limiting step as well.

Multiple Effects of Cholesterol on Influenza Membrane Fusion Kinetics

Influenza and other enveloped viruses differ in their envelope lipid composition from the cellular plasma membrane from which they bud. Viruses also appear to fuse preferentially to specific membrane compartments, suggesting that the lipid environment may play a selective role in permissiveness for fusion. Cholesterol in particular is enriched in the viral membrane and known to exert effects on fusion kinetics and efficiency in a number of model systems for viral infection. Cholesterol can affect membranes in multiple ways, both impacting materials properties such as membrane bending energy and lateral organization of the membrane. To probe the different effects of cholesterol, we have measured viral fusion kinetics as we vary the cholesterol concentration in both live influenza virus and target liposomes. The liposomal membrane composition was selected not to support lateral membrane heterogeneity. We observe an increase in fusion rate with increasing liposomal cholesterol concentration, consistent with a model where cholesterol promotes stalk formation and stalk formation is the rate-limiting step of the fusion reaction we measure. We observe a more complex dose-response relationship as cholesterol is extracted from influenza virions. Since hemagglutinin is known to have cholesterol-dependent lateral organization, we postulate that the altered kinetics with changes to viral sterol content reflect both changes to hemagglutinin organization in the membrane and changes to the material properties of the membranes. We further characterize this dose-response relationship to help resolve these disparate effects of cholesterol in mediating viral entry.

Kinetic Mechanism of Indole-3-glycerol Phosphate Synthase

The (βα)(8)-barrel enzyme indole-3-glycerol phosphate synthase (IGPS) catalyzes the multistep transformation of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) into indole-3-glycerol phosphate (IGP) in tryptophan biosynthesis. Mutagenesis data and crystal structure analysis of IGPS from Sulfolobus solfataricus (sIGPS) allowed for the formulation of a plausible chemical mechanism of the reaction, and molecular dynamics simulations suggested that flexibility of active site loops might be important for catalysis. Here we developed a method that uses extrinsic fluorophores attached to active site loops to connect the kinetic mechanism of sIGPS to structure and conformational motions. Specifically, we elucidated the kinetic mechanism of sIGPS and correlated individual steps in the mechanism to conformational motions of flexible loops. Pre-steady-state kinetic measurements of CdRP to IGP conversion monitoring changes in intrinsic tryptophan and IGP fluorescence provided a minimal three-step kinetic model in which fast substrate binding and chemical transformation are followed by slow product release. The role of sIGPS loop conformational motion during substrate binding and catalysis was examined via variants that were covalently labeled with fluorescent dyes at the N-terminal extension of the enzyme and mobile active site loop β1α1. Analysis of kinetic data monitoring dye fluorescence revealed a conformational change that follows substrate binding, suggesting an induced-fit-type binding mechanism for the substrate CdRP. Global fitting of all kinetic results obtained with wild-type sIGPS and the labeled variants was best accommodated by a four-step kinetic model. In this model, both the binding of CdRP and its on-enzyme conversion to IGP are accompanied by conformational transitions. The liberation of the product from the active site is the rate-limiting step of the overall reaction. Our results confirm the importance of flexible active loops for substrate binding and catalysis by sIGPS.

Characterization of the Double Perovskite Ba2BixSc0.2Co1.8–xO6−δ (x = 0.1, 0.2)

The Ba2BixSc0.2Co1.8–xO6−δ (x = 0.1 or 0.2) cubic perovskites are confirmed to be mixed-valent Co3+/Co2+ oxides that, as mixed oxide-ion and electronic conductors, are superior cathode materials for an intermediate-temperature solid oxide fuel cell catalyzing the oxygen-reduction reaction. Neutron powder diffraction for x = 0.2 was used to confirm also that these compounds have the double-perovskite structure Ba2BB′O6−δ at room temperature with B = Co, B′ = Bi0.2Sc0.2Co0.6, δ ≈ 1.3, and a B–O bond length a little longer than the mean B′–O bond length. These data indicate that the room-temperature x = 0.2 phase is Ba2Co2+B′O4.7 with B′ = (Bi5+)0.2(Sc3+)0.2(Co3+)0.6. The presence of Bi5+ was confirmed by comparing the Bi4f core-level X-ray photoelectron spectrum of the x = 0.2 sample with that of BaBiO3. BaCoO3 crystallizes in the 2H hexagonal polytype as a result of t ≡ (A–O)/[√2(B–O)] > 1, where A–O and B–O are the equilibrium bond lengths of an ABO3 perovskite. Substitution of Bi and Sc for Co as well as the incorporation of oxygen vacancies increases the mean B–O bond length to reduce t > 1 to where cubic stacking of close-packed BaO3 planes is energetically favored over hexagonal stacking that introduces large B-cation Coulombic repulsions across a shared octahedral-site face. Cooperative oxygen displacements that create larger B–O than B′–O mean bond length are characteristic of oxide ions in a cubic perovskite with geometric tolerance factor t > 1 as illustrated by ferroelectric BaTiO3. Disordering of the double-perovskite oxide-ion displacements occurs over the range 350–400 °C; long-range, frustrated magnetic order occurs below a Tc ≈ 35 K. X-ray absorption spectroscopy measurements show the absence of low-spin Co3+ at room temperature, and the rate-limiting step of the oxygen-reduction reaction on these oxides acting as cathodes of a solid oxide fuel cell is shown to be electron transfer from the oxide to an adsorbed O2, which we conjecture occurs at surface intermediate-spin Co3+ ions.

Study of NO oxidation reaction over the Pt cluster supported on γ-Al2O3(111) surface

NO oxidation reaction on the Pt cluster supported on γ-Al2O3(111) surface is investigated using the climbing image nudged elastic band (CI-NEB) calculation based on the density functional theory. This is to verify the possibility of using Pt-based catalyst for NO oxidation reaction which is the rate-limiting step for the NOx oxidation reaction. From investigations of NO adsorption and NO2 desorption on the Pt cluster supported on γ-Al2O3 (111) surface where the dissociated O atoms already formed and lie on the Pt cluster sites, we find the process requires no activation barrier. Furthermore, we also performed the first principles of molecular dynamics simulation to investigate NO oxidation reaction at certain range temperatures (400 K, 600 K and 800 K) for complement of CI-NEB calculation. From the results, we discussed the temperature dependence of NO oxidation reaction.

Corrosion kinetics under high pressure of steam of pure zirconium and zirconium alloys followed by in situ thermogravimetry

A new experimental thermogravimetric device has been installed to study in situ the corrosion behaviour of zirconium alloys under high pressure of steam. Corrosion tests up to 5MPa of steam pressure have been performed on two materials, pure zirconium and Zircaloy-4 (Zy4), at around 415°C. The rate-limiting step assumption was experimentally verified on Zy4. Unlike pure zirconium, its oxidation rate is not dependent on steam pressure. The experimental result obtained on this material is consistent with an oxygen vacancy diffusion rate-limiting step. For pure zirconium, the kinetic law is nearly linear during the corrosion process, which leads to propose an interface reaction rate-limiting step. Moreover, according to the isotope exchange experiments, the oxygen diffusion in the oxide formed on pure zirconium under high pressure of steam is very fast compared to that in the oxide of Zy4, which supports the thermogravimetric results. Finally, the impact of the SPPs on the corrosion resistance is briefly discussed in the last part of this paper from photoelectrochemical results.

Accurate Rate Constants for Decomposition of Aqueous Nitrous Acid

Decomposition of nitrous acid in aqueous solution has been studied by stopped flow spectrophotometry to resolve discrepancies in literature values for the rate constants of the decomposition reactions. Under the conditions employed, the rate-limiting reaction step comprises the hydrolysis of NO(2). A simplified rate law based on the known elementary reaction mechanism provides an excellent fit to the experimental data. The rate constant, 1.34 × 10(-6) M(-1) s(-1), is thought to be of higher accuracy than those in the literature as it does not depend on the rate of parallel reaction pathways or on the rate of interphase mass transfer of gaseous reaction products. The activation energy for the simplified rate law was established to be 107 kJ mol(-1). Quantum chemistry calculations indicate that the majority of the large activation energy results from the endothermic nature of the equilibrium 2HNO(2) ⇆ NO + NO(2) + H(2)O. The rate constant for the reaction between nitrate ions and nitrous acid, which inhibits HNO(2) decomposition, was also determined.

Pre-Steady-State Kinetic Characterization of Thiolate Anion Formation in Human Leukotriene C4 Synthase

Human leukotriene C₄ synthase (hLTC4S) is an integral membrane protein that catalyzes the committed step in the biosynthesis of cysteinyl-leukotrienes, i.e., formation of leukotriene C₄ (LTC₄). This molecule, together with its metabolites LTD₄ and LTE₄, induces inflammatory responses, particularly in asthma, and thus, the enzyme is an attractive drug target. During the catalytic cycle, glutathione (GSH) is activated by hLTC4S that forms a nucleophilic thiolate anion that will attack LTA₄, presumably according to an S(N)2 reaction to form LTC₄. We observed that GSH thiolate anion formation is rapid and occurs at all three monomers of the homotrimer and is concomitant with stoichiometric release of protons to the medium. The pK(a) (5.9) for enzyme-bound GSH thiol and the rate of thiolate formation were determined (k(obs) = 200 s⁻¹). Taking advantage of a strong competitive inhibitor, glutathionesulfonic acid, shown here by crystallography to bind in the same location as GSH, we determined the overall dissociation constant (K(d((GS) = 14.3 μM). The release of the thiolate was assessed using a GSH release experiment (1.3 s⁻¹). Taken together, these data establish that thiolate anion formation in hLTC4S is not the rate-limiting step for the overall reaction of LTC₄ production (k(cat) = 26 s⁻¹), and compared to the related microsomal glutathione transferase 1, which displays very slow GSH thiolate anion formation and one-third of the sites reactivity, hLTC4S has evolved a different catalytic mechanism.