Kinetic analysis of the slow ionization of glutathione by microsomal glutathione transferase MGST1

An important aspect of the catalytic mechanism of microsomal glutathione transferase (MGST1) is the activation of the thiol of bound glutathione (GSH). GSH binding to MGST1 as measured by thiolate anion formation, proton release, and Meisenheimer complex formation is a slow process that can be described by a rapid binding step (K(GSH)d = 47 +/- 7 mM) of the peptide followed by slow deprotonation (k2 = 0.42 +/- 0.03 s(-1). Release of the GSH thiolate anion is very slow (apparent first-order rate k(-2) = 0.0006 +/- 0.00002 s(-)(1)) and thus explains the overall tight binding of GSH. It has been known for some time that the turnover (kcat) of MGST1 does not correlate well with the chemical reactivity of the electrophilic substrate. The steady-state kinetic parameters determined for GSH and 1-chloro-2,4-dinitrobenzene (CDNB) are consistent with thiolate anion formation (k2) being largely rate-determining in enzyme turnover (kcat = 0.26 +/- 0.07 s(-1). Thus, the chemical step of thiolate addition is not rate-limiting and can be studied as a burst of product formation on reaction of halo-nitroarene electrophiles with the E.GS- complex. The saturation behavior of the concentration dependence of the product burst with CDNB indicates that the reaction occurs in a two-step process that is characterized by rapid equilibrium binding ( = 0.53 +/- 0.08 mM) to the E.GS- complex and a relatively fast chemical reaction with the thiolate (k3 = 500 +/- 40 s(-1). In a series of substrate analogues, it is observed that log k3 is linearly related (rho value 3.5 +/- 0.3) to second substrate reactivity as described by Hammett sigma- values demonstrating a strong dependence on chemical reactivity that is similar to the nonenzymatic reaction (rho = 3.4). Microsomal glutathione transferase 1 displays the unusual property of being activated by sulfhydryl reagents. When the enzyme is activated by N-ethylmaleimide, the rate of thiolate anion formation is greatly enhanced, demonstrating for the first time the specific step that is activated. This result explains earlier observations that the enzyme is activated only with more reactive substrates. Taken together, the observations show that the kinetic mechanism of MGST1 can be described by slow GSH binding/thiolate formation followed by a chemical step that depends on the reactivity of the electrophilic substrate. As the chemical reactivity of the electrophile becomes lower the rate-determining step shifts from thiolate formation to the chemical reaction.     

Ligand-induced changes in the structure and dynamics of a human class Mu glutathione S-transferase

Glutathione transferases are detoxification enzymes that catalyze the addition of glutathione (GSH) to a wide variety of hydrophobic compounds. Although this group of enzymes has been extensively characterized by crystallographic studies, little is known about their dynamic properties. This study investigates the role of protein dynamics in the mechanism of a human class mu enzyme (GSTM2-2) by characterizing the motional properties of the unliganded enzyme, the enzyme-substrate (GSH) complex, an enzyme-product complex [S-(2,4-dinitrobenzyl)glutathione, GSDNB], and an enzyme-inhibitor complex (S-1-hexylglutathione, GSHEX). The kinetic on- and off-rates for these ligands are 10-20-fold lower than the diffusion limit, suggesting dynamic conformational heterogeneity of the active site. The off-rate of GSDNB is similar to the turnover number for its enzymatic formation, suggesting that product release is rate-limiting when 1-chloro-2,4-dinitrobenzene is the substrate. The dynamic properties of GSTM2-2 were investigated over a wide range of time scales using (15)N nuclear spin relaxation, residual dipolar couplings, and amide hydrogen-deuterium exchange rates. These data show that the majority of the protein backbone is rigid on the nanosecond to picosecond time scale for all forms of the enzyme. The presence of motion on the millisecond to microsecond time scale was detected for a small number of residues within the active site. These motions are likely to play a role in facilitating substrate binding and product release. The residual dipolar couplings also show that the conformation of the active site region is more open in solution than in the crystalline environment, further enhancing ligand accessibility to the active site. Amide hydrogen-deuterium exchange rates indicate a reduction in the dynamic properties of several residues near the active site due to the binding of ligand. GSH binding reduces the exchange rate of a number of residues in proximity to its binding site, while GSHEX causes a reduction in amide-exchange rates throughout the entire active site region. The location of the dinitrobenzene (DNB) ring in the GSDNB-GSTM2-2 complex was modeled using chemical shift changes that occur when GSDNB binds to the enzyme. The DNB ring makes a number of contacts with hydrophobic residues in the active site, including Met108. Replacement of Met108 with Ala increases the turnover number of the enzyme by a factor of 1.7.     

Dichloromethane dehalogenase of Hyphomicrobium sp. strain DM2.

Dichloromethane dehalogenase, a highly inducible glutathione-dependent enzyme catalyzing the conversion of dichloromethane into formaldehyde and inorganic chloride, was purified fivefold with 60% yield from Hyphomicrobium sp. strain DM2. The electrophoretically homogeneous purified enzyme exhibited a specific activity of 17.3 mkat/kg of protein. Its pH optimum was 8.5. The enzyme was stable at -20 degrees C for at least 6 months. A subunit molecular weight of 33,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration of native dichloromethane dehalogenase yielded a molecular weight of 195,000. Subunit cross-linking with dimethyl suberimidate confirmed the hexameric tertiary structure of the enzyme. Dichloromethane dehalogenase was highly specific for dihalomethanes. Its apparent Km values were 30 microM for CH2Cl2, 15 microM for CH2BrCl, 13 microM for CH2Br2, 5 microM for CH2I2, and 320 microM for glutathione. Several chlorinated aliphatic compounds inhibited the dichloromethane dehalogenase activity of the pure enzyme. The Ki values of the competitive inhibitors 1,2-dichloroethane and 1-chloropropane were 3 and 56 microM, respectively.     

Genetic control of methyl halide production in Arabidopsis

Methyl chloride (CH(3)Cl) and methyl bromide (CH(3)Br) are the primary carriers of natural chlorine and bromine, respectively, to the stratosphere, where they catalyze the destruction of ozone, whereas methyl iodide (CH(3)I) influences aerosol formation and ozone loss in the boundary layer. CH(3)Br is also an agricultural pesticide whose use is regulated by international agreement. Despite the economic and environmental importance of these methyl halides, their natural sources and biological production mechanisms are poorly understood. Besides CH(3)Br fumigation, important sources include oceans, biomass burning, tropical plants, salt marshes, and certain crops and fungi. Here, we demonstrate that the model plant Arabidopsis thaliana produces and emits methyl halides and that the enzyme primarily responsible for the production is encoded by the HARMLESS TO OZONE LAYER (HOL) gene. The encoded protein belongs to a group of methyltransferases capable of catalyzing the S-adenosyl-L-methionine (SAM)-dependent methylation of chloride (Cl(-)), bromide (Br(-)), and iodide (I(-)) to produce methyl halides. In mutant plants with the HOL gene disrupted, methyl halide production is largely eliminated. A phylogenetic analysis with the HOL gene suggests that the ability to produce methyl halides is widespread among vascular plants. This approach provides a genetic basis for understanding and predicting patterns of methyl halide production by plants.     

Glutathione activation of chloropicrin in the Salmonella mutagenicity test

Chloropicrin (CCl3NO2) is a major soil fumigant for control of fungi, insects and nematodes and may by formed by chlorination of drinking water. It is also a strong lacrimator and induces sister chromatid exchanges in cultured human lymphocytes. Mutagenicity assays of CCl3NO2 in Salmonella typhimurium TA100 establish that it is toxic but not mutagenic at 500 nmol/plate but becomes mutagenic but not toxic on addition of S9 (previous work) or 1-2 molar equivalents of glutathione (GSH) (this study). The preincubation assay is superior to the plate incorporation test giving 2-4-fold higher revertants/nmol. Using the preincubation assay with GSH at 5 mM (a biomimetic level) in the top agar gives linear dose-response relationships for CCl3NO2 and its dechlorination products CHCl2NO2 and CH2ClNO2 with 0.56, 0.56 and 1.8 revertants/nmol, respectively. The mutagenicity values for CHCl2NO2 and CH2ClNO2 are the same in the presence and the absence of GSH, which only improves the linearity at high levels by reducing toxicity to the bacteria. GSH activation of CCl3NO2 mutagenicity may be due to reductive dechlorination of the trichloro compound to the more active CHCl2NO2 and CH2ClNO2. Alternatively, the mutagenicity may result from an intermediate GSH conjugate such as GSCCl2NO2 or GSCHClNO2. In comparison, the mutagenicity of CH2Br2 and CH2I2 is affected little if any by addition of GSH and these dihalomethanes are much less active than the halonitromethane series. It therefore appears that CCl3NO2 is not mutagenic in the absence of activation and that the dechlorinated metabolites CHCl2NO2 and CH2ClNO2 are moderately potent bacterial mutagens, consistent with the possible genotoxicity of CCl3NO2 in mammals.     

Halomethane:bisulfide/halide ion methyltransferase, an unusual corrinoid enzyme of environmental significance isolated from an aerobic methylotroph using chloromethane as the sole carbon source.

A novel dehalogenating/transhalogenating enzyme, halomethane:bisulfide/halide ion methyltransferase, has been isolated from the facultatively methylotrophic bacterium strain CC495, which uses chloromethane (CH(3)Cl) as the sole carbon source. Purification of the enzyme to homogeneity was achieved in high yield by anion-exchange chromatography and gel filtration. The methyltransferase was composed of a 67-kDa protein with a corrinoid-bound cobalt atom. The purified enzyme was inactive but was activated by preincubation with 5 mM dithiothreitol and 0.5 mM CH(3)Cl; then it catalyzed methyl transfer from CH(3)Cl, CH(3)Br, or CH(3)I to the following acceptor ions (in order of decreasing efficacy): I(-), HS(-), Cl(-), Br(-), NO(2)(-), CN(-), and SCN(-). Spectral analysis indicated that cobalt in the native enzyme existed as cob(II)alamin, which upon activation was reduced to the cob(I)alamin state and then was oxidized to methyl cob(III)alamin. During catalysis, the enzyme shuttles between the methyl cob(III)alamin and cob(I)alamin states, being alternately demethylated by the acceptor ion and remethylated by halomethane. Mechanistically the methyltransferase shows features in common with cobalamin-dependent methionine synthase from Escherichia coli. However, the failure of specific inhibitors of methionine synthase such as propyl iodide, N(2)O, and Hg(2+) to affect the methyltransferase suggests significant differences. During CH(3)Cl degradation by strain CC495, the physiological acceptor ion for the enzyme is probably HS(-), a hypothesis supported by the detection in cell extracts of methanethiol oxidase and formaldehyde dehydrogenase activities which provide a metabolic route to formate. 16S rRNA sequence analysis indicated that strain CC495 clusters with Rhizobium spp. in the alpha subdivision of the Proteobacteria and is closely related to strain IMB-1, a recently isolated CH(3)Br-degrading bacterium (T. L. Connell Hancock, A. M. Costello, M. E. Lidstrom, and R. S. Oremland, Appl. Environ. Microbiol. 64:2899-2905, 1998). The presence of this methyltransferase in bacterial populations in soil and sediments, if widespread, has important environmental implications.     

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of the catalytic activity of glutathione transferases

Steady state, pre-steady state kinetic experiments, and site-directed mutagenesis have been used to dissect the catalytic mechanism of human glutathione transferase T2-2 with 1-menaphthyl sulfate as co-substrate. This enzyme is close to the ancestral precursor of the more recently evolved glutathione transferases belonging to Alpha, Pi, and Mu classes. The enzyme displays a random kinetic mechanism with very low k(cat) and k(cat)/K(m)((GSH)) values and with a rate-limiting step identified as the product release. The chemical step, which is fast and causes product accumulation before the steady state catalysis, strictly depends on the deprotonation of the bound GSH. Replacement of Arg-107 with Ala dramatically affects the fast phase, indicating that this residue is crucial both in the activation and orientation of GSH in the ternary complex. All pre-steady state and steady state kinetic data were convincingly fit to a kinetic mechanism that reflects a quite primordial catalytic efficiency of this enzyme. It involves two slowly interconverting or not interconverting enzyme populations (or active sites of the dimeric enzyme) both able to bind and activate GSH and strongly inhibited by the product. Only one population or subunit is catalytically competent. The proposed mechanism accounts for the apparent half-site behavior of this enzyme and for the apparent negative cooperativity observed under steady state conditions. These findings also suggest some evolutionary strategies in the glutathione transferase family that have been adopted for the optimization of the catalytic activity, which are mainly based on an increased flexibility of critical protein segments and on an optimal orientation of the substrate.     

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.     

The permeability of hydrophobic membranes to 22Na salts and 14CO2 in low dielectric media

The one-way fluxes of 14CO2 and a series of 22Na (Cl, Br, HCO3, ClO4, I) salts across n-hexadecane-impregnated solid-support liquid membranes have been measured in water and low dielectric media (50-90 vol% dioxane/water). One-way fluxes for 14CO2 (J14CO2) were 0.84 and 1.03 x 10(-9) mol cm-2 s-1 in 75% dioxane (aq.) and water, respectively, across both impregnated cellulose and teflon membranes. 22Na fluxes across impregnated cellulose membranes in 75% dioxane (aq.) ranged from 1.8 to 11.4 x 10(-10) mol cm-2 s-1 and had the order NaCl less than NaBr less than NaHCO3 less than NaClO4 less than Nal. 22Na fluxes across impregnated teflon membranes were slightly smaller, 1.5-7.1 x 10(-10) mol cm-2 s-1, but had the same order for the anions tested. No measurable 22Na fluxes were observed in aqueous media. For NaI and NaClO4 there was a 3-6-fold enhancement of fluxes in 90% dioxane (aq.) compared to 75% dioxane (aq.). The corresponding enhancement for fluxes of NaHCO3, NaBr and NaCl was 1.5-fold. The results are discussed in terms of ion-paired salt transport in low dielectric media.     

Pre-steady-state kinetic characterization of thiolate anion formation in human leukotriene C₄ synthase

Human leukotriene C? synthase (hLTC4S) is an integral membrane protein that catalyzes the committed step in the biosynthesis of cysteinyl-leukotrienes, i.e., formation of leukotriene C? (LTC?). This molecule, together with its metabolites LTD? and LTE?, 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 LTA?, presumably according to an S(N)2 reaction to form LTC?. 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 LTC? 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.     

Kinetic mechanism of choline oxidase from Arthrobacter globiformis

Choline oxidase catalyzes the four-electron oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate and molecular oxygen as primary electron acceptor. The enzyme is capable of accepting betaine-aldehyde as a substrate, allowing the investigation of the reaction mechanism for both the conversion of choline to the aldehyde intermediate and of betaine-aldehyde to glycine-betaine. The steady state kinetic mechanism has been determined at pH 7 with choline and betaine-aldehyde as substrate to be sequential, consistent with oxygen reacting with the reduced enzyme before release of betaine-aldehyde or glycine-betaine, respectively. A K(m) value < or =20 microM has been estimated for betaine-aldehyde based on the kinetic pattern with a y-intercept seen in a plot of 1/rate versus 1/[oxygen]. The kinetic data suggest that betaine-aldehyde predominantly remains bound at the active site during turnover of the enzyme with choline. In agreement with such a conclusion, less than 10% betaine-aldehyde has been found in the reaction mixture under enzymatic turnover with saturating concentrations of choline. The k(cat) values were 6.4+/-0.3 and 15.3+/-2.5 s(-1) for choline and betaine-aldehyde, respectively, suggesting that a kinetic step in the oxidation of choline to the aldehyde intermediate must be partially rate-limiting for catalysis. Cleavage of the CH bond of choline as being partially rate-limiting for catalysis is discussed.     

Stereoselectivity and enantioselectivity of glutathione S-transferase toward stilbene oxide substrates

Isozyme 4-4 of rat liver glutathione S-transferase catalyzes the stereoselective addition of glutathione to the oxirane carbon of R-absolute configuration of cis-stilbene oxide, 2, to give 98 +/- 2% of the (1S,2S)-1,2-diphenyl-1-(S-glutathionyl)-2-hydroxyethane product with a turnover number (kc) of 0.22 s-1. The two enantiomers of trans-stilbene oxide, 3, are somewhat poorer substrates for the enzyme. Enantioselective addition of glutathione to 3 proceeds with turnover numbers of 0.12 s-1 and 0.023 s-1 for the (R,R,)- and (S,S)-antipodes, respectively.     

Induction of DNA strand breaks by trihalomethanes in primary human lung epithelial cells

Trihalomethanes (THMs) are disinfection by-products and suspected human carcinogens present in chlorinated drinking water. Previous studies have shown that many THMs induce sister chromatid exchanges and DNA strand breaks in human peripheral blood lymphocytes in vitro. Exposure to THMs occurs through oral, dermal, or inhalation routes, with the lung being a target of exposure by the latter route, although not a target for rodent carcinogenicity. Thus, to examine the genotoxicity of THMs in this tissue, we used the comet assay to examine the DNA damaging ability of five THMs in primary human lung epithelial cells. Cells were collected by scraping the large airways of four volunteers with a cytology brush and then passaging the cells no more than three times in order to have sufficient numbers for the experiments. Cells were exposed for 3h to 10, 100, or 1000 microM CHCl(3), CHCl(2)Br, CHClBr(2), or CHBr(3); CH(2)Cl(2) was also used as a model dihalomethane for comparison to the THMs. The compounds ranked as follows for DNA damaging ability: CHCl(2)Br>CHBr(3)>CHCl(3) approximately equal CH(2)Cl(2); CHClBr(2) was negative. Considerable inter-individual variation was observed. For example, CHCl(3) was genotoxic in only two subjects, and the interaction between dose and donor was highly significant (P<0.001). The same variation was observed for CHCl(2)Br, which was positive only in the two subjects in which CHCl(3) was negative. This variation was not due to the GSTT1-1 genotype of the subjects. Although two subjects were GSTT1-1(+), and two were GSTT1-1(-), no cultured cells with a GSTT1-1(+) genotype had detectable GSTT1-1 enzymatic activity nor did any frozen epithelial cells that had not been cultured. However, GSTT1-1 enzymatic activity was detected in fresh (neither frozen nor cultured) lung cells. These results show that freezing or culturing causes lung cells to lose GSTT1-1 activity and that factors other than GSTT1-1 activity play a role in the variable responses of these human cells to the genotoxicity of the halomethanes studied here.     

2-Hydroxychromene-2-carboxylic acid isomerase: a kappa class glutathione transferase from Pseudomonas putida

The enzyme 2-hydroxychromene-2-carboxylic acid (HCCA) isomerase catalyzes the glutathione (GSH)-dependent interconversion (Keq = 1.5) of HCCA and trans-o-hydroxybenzylidene pyruvic acid (tHBPA) in the naphthalene catabolic pathway of Pseudomonas putida. The dimeric protein binds one molecule of GSH very tightly (Kd approximately 5 nM) and a second molecule of GSH with much lower affinity (Kd approximately 2 to 11 microM). The enzyme is unstable in the absence of GSH. The turnover number in the forward direction (47 s(-1) at 25 degrees C) greatly exceeds off rates for GSH (koff approximately 10(-3) to 10(-2) s(-1) at 10 degrees C), suggesting that GSH acts as a tightly bound cofactor in the reaction. The crystal structure of the enzyme at 1.7 A resolution reveals that the isomerase is closely related to class kappa GSH transferases. Diffraction quality crystals could only be obtained in the presence of GSH and HCCA/tHBPA. Clear electron density is seen for GSH. Electron density for the organic substrates is located near the GSH and is best modeled to include both HCCA and tHBPA at occupancies of 0.5 for each. Although there is no electron density connecting the sulfur of GSH to the organic substrates, the sulfur is located very close (2.78 A) to C7 of HCCA. Taken together, the results suggest that the isomerization reaction involves a short-lived covalent adduct between the sulfur of GSH and C7 of the substrate.     

Transient-state kinetic analysis of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase reveals that a step after chemical transformation is rate limiting

MyristoylCoA:protein N-myristoyltransferase is a member of the superfamily of GCN5-related N-acetyltransferases and catalyzes the covalent attachment of myristate to the N-terminal Gly residue of proteins with diverse functions. Saccharomyces cerevisiae Nmt1p is a monomeric protein with an ordered bi-bi reaction mechanism: myristoylCoA is bound prior to peptide substrate; after catalysis, CoA is released followed by myristoylpeptide. Analysis of the X-ray structure of Nmt1p with bound substrate analogues indicates that the active site contains an oxyanion hole and a catalytic base and that catalysis proceeds through the nucleophilic addition-elimination mechanism. To determine the rate-limiting step in the enzyme reaction, pre-steady-state kinetic analyses were performed using a new, sensitive nonradioactive assay that detects CoA. Multiple turnover quenched flow studies disclosed that a step after the chemical transformation limits the overall rate of the reaction. Multiple and single turnover analyses revealed that the rate for the chemical transformation step is 13.8+/-0.6 s(-1) while the slower steady-state phase is 0.10+/-0.01 s(-1). Stopped flow kinetic studies of substrate acquisition indicated that binding of myristoylCoA to the apo-enzyme occurs through at least a two-step process, with a fast phase rate of 3.2 x 10(8) M(-1) s(-1) and a slow phase rate of 23+/-2 s(-1) (defined at 5 degrees C). Binding of an octapeptide substrate, representing the N-terminal sequence of a known yeast N-myristoylprotein (Cnb1p), to a binary complex composed of Nmt1p and a nonhydrolyzable myristoylCoA analogue (S-(2-oxo)pentadecylCoA) has a second-order rate constant of 2.1+/-0.3 x 10(6) M(-1) s(-1) and a dissociation rate of 26+/-15 s(-1) (defined at 10 degrees C). These results are interpreted in light of the X-ray structures of this enzyme.     

Parallel evolutionary pathways for glutathione transferases: structure and mechanism of the mitochondrial class kappa enzyme rGSTK1-1

The class kappa glutathione (GSH) transferase is an enzyme that resides in the mitochondrial matrix. Its relationship to members of the canonical GSH transferase superfamily has remained an enigma. The three-dimensional structure of the class kappa enzyme from rat (rGSTK1-1) in complex with GSH has been solved by single isomorphous replacement with anomalous scattering at a resolution of 2.5 A. The structure reveals that the enzyme is more closely related to the protein disulfide bond isomerase, dsbA, from Escherichia coli than it is to members of the canonical superfamily. The structures of rGSTK1-1 and the canonical superfamily members indicate that the proteins folds have diverged from a common thioredoxin/glutaredoxin progenitor but did so by different mechanisms. The mitochondrial enzyme, therefore, represents a fourth protein superfamily that supports GSH transferase activity. The thioredoxin domain functions in a manner that is similar to that seen in the canonical enzymes by providing key structural elements for the recognition of GSH. The hydroxyl group of S16 is within hydrogen-bonding distance of the sulfur of bound GSH and is, in part, responsible for the ionization of the thiol in the E*GSH complex (pKa = 6.4 +/- 0.1). Preequilibrium kinetic experiments indicate that the k(on) for GSH is 1 x 10(5) M(-1) s(-1) and k(off) for GS- is approximately 8 s(-1) and relatively slow with respect to turnover with 1-chloro-2, 4-dinitrobenzene (CDNB). As a result, the KM(GSH) (11 mM) is much larger than the apparent Kd(GSH) (90 microM). The active site has a relatively open access channel that is flanked by disordered loops that may explain the relatively high turnover number (280 s(-1) at pH 7.0) toward CDNB. The disordered loops form an extensive contiguous patch on one face of the dimeric enzyme, a fact that suggests that the protein surface may interact with a membrane or other protein partner.     

Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when homozygous, and proposed model for moderate glutathione deficiency when heterozygous

The biosynthesis of reduced glutathione (GSH) is carried out by the enzymes gamma-glutamylcysteine synthetase (GCL) and GSH synthetase. GCL is the rate-limiting step and represents a heterodimeric enzyme comprised of a catalytic subunit (GCLC) and a ("regulatory"), or modifier, subunit (GCLM). The nonhomologous Gclc and Gclm genes are located on mouse chromosomes 9 and 3, respectively. GCLC owns the catalytic activity, whereas GCLM enhances the enzyme activity by lowering the K(m) for glutamate and increasing the K(i) to GSH inhibition. Humans have been identified with one or two defective GCLC alleles and show low GSH levels. As an initial first step toward understanding the role of GSH in cellular redox homeostasis, we have targeted a disruption of the mouse Gclc gene. The Gclc(-/-) homozygous knockout animal dies before gestational day 13, whereas the Gclc(+/-) heterozygote is viable and fertile. The Gclc(+/-) mouse exhibits a gene-dose decrease in the GCLC protein and GCL activity, but only about a 20% diminution in GSH levels and a compensatory increase of approximately 30% in ascorbate-as compared with that in Gclc(+/+) wild-type littermates. These data show a reciprocal action between falling GSH concentrations and rising ascorbate levels. Therefore, the Gclc(+/-) mouse may be a useful genetic model for mild endogenous oxidative stress.     

A stopped flow transient kinetic analysis of substrate binding and catalysis in Escherichia coli D-3-phosphoglycerate dehydrogenase

Pre-steady state, stopped flow analysis of Escherichia coli D-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior. NADH binding was consistent with an initial binding event followed by a slow conformational change for each site. The slow conformational change is responsible for the apparent tight binding of NADH to the apoenzyme but is too slow to participate in the catalytic cycle when the enzyme is rapidly turning over. Subsequent binding of the substrate, alpha-ketoglutarate, was characterized by a rapid equilibrium binding event followed by a conformational change for each site. Catalysis in the direction of NAD(+) reduction showed a distinct burst of activity followed by a slow rate of turnover, indicating that the rate-limiting step is after hydride transfer. Catalysis in the direction of NADH oxidation did not display burst kinetics, indicating that the rate-limiting step is at or before the hydride transfer step. The burst data indicated that the rate of NAD(+) reduction (3.8 s(-1)) is similar to the k(cat) of the enzyme (2-3 s(-1)) in that direction. However, analysis of the reaction with deuterated NADH failed to show an effect on the velocity of the reaction with a V(H)/V(D)=1.07+/-0.06. None of the other rates determined by stopped flow analysis could account for the k(cat) of the enzyme in either direction (forward k(cat)=0.01 s(-1), reverse k(cat)=2-3 s(-1)), suggesting that the rate-limiting step in both directions is a conformational change in the enzyme that is not detected optically.     

Structural basis and mechanism of the inhibition of the type-3 copper protein tyrosinase from Streptomyces antibioticus by halide ions

The inhibition of the type-3 copper enzyme tyrosinase by halide ions was studied by kinetic and paramagnetic (1)H NMR methods. All halides are inhibitors in the conversion of l-3,4-dihydroxyphenylalanine (l-DOPA) with apparent inhibition constants that follow the order I(-) < F(-) < Cl(-) < Br(-) at pH 6.80. The results show that the inhibition arises from the interaction of halide with both the oxidized (affinity F(-) > Cl(-) > Br(-) > I(-)) and reduced (affinity I(-) > Br(-) > Cl(-) > F(-)) enzyme. The paramagnetic (1)H NMR of the oxidized enzyme complexed with the halides is consistent with a direct interaction of halide with the type-3 site and shows that the (Cu-His(3))(2) coordination occurs in all halide-bound species. It is surmised that halides bridge both of the copper ions in the active site. Fluoride and chloride are shown to bind only to the low pH form of oxidized tyrosinase, explaining the strong pH dependence of the inhibition by these ions. We further show that p-toluic acid and the bidentate transition state analogue, Kojic acid, displace chloride from the oxidized active site, whereas the monodentate substrate analogue, p-nitrophenol, forms a ternary complex with the enzyme and the chloride ion. On the basis of the experimental results, a model is formulated for the inhibitor action and for the reaction of diphenols with the oxidized enzyme.     

Kinetic characterization of thiolate anion formation and chemical catalysis of activated microsomal glutathione transferase 1

Microsomal glutathione transferase 1 (MGST1) displays the unique ability to be activated, up to 30-fold, by the reaction with sulfhydryl reagents, e.g., N-ethylmaleimide. Analysis of glutathione (GSH) thiolate formation, which occurs upon mixing activated MGST1 with GSH, reveals biphasic kinetics, where the rapid phase dominated at higher GSH concentrations. The kinetic behavior suggests a two-step mechanism consisting of a rapid GSH-binding step (K(D)(GSH) approximately 10 mM), followed by slower formation of thiolate (k(2) approximately 10 s(-1)). The release rate (or protonation of the enzyme GSH thiolate complex) of GS(-) was slow (k(-2) = 0.016 s(-1)), consistent with overall tight binding of GSH. Electrophilic second substrates react rapidly with the E*GS(-) complex, and again, a two-step mechanism is suggested. In comparison to the unactivated enzyme [Morgenstern et al. (2001) Biochemistry 40, 3378-3384], the mechanisms of GSH thiolate formation and electrophile interaction are similar; however, thiolate anion formation is enhanced 30-fold in the activated enzyme, contributing to an increased k(cat) (3.6 s(-1)). Interestingly, in the activated enzyme, thiolate formation and proton release from the enzyme are not strictly coupled, because proton release (as well as k(cat)) was found to be approximately 4 times slower than GSH thiolate formation in an unbuffered system. Solvent kinetic isotope effect measurements demonstrated a 2-fold decrease in the rate constant (k(2)) for thiolate formation and k(cat) (in the reaction with 1-chloro-2,4-dinitrobenzene) for both unactivated and activated MGST1. This indicates that thiolate formation contributes to k(cat) for the activated enzyme, as suggested previously for unactivated MGST1. The stoichiometry of thiolate formation, proton release, and burst kinetics suggested utilization of one GSH molecule per enzyme trimer.