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W2079548047 abstract "The homodimeric flavoenzyme glutathione reductase (GR) maintains high intracellular concentrations of the antioxidant glutathione (GSSG + NADPH + H+ ↔ 2 GSH + NADP+). Due to its central function in cellular redox metabolism, inhibition of GR from the malarial parasitePlasmodium falciparum represents an important approach to antimalarial drug development; therefore, the catalytic mechanism of GR from P. falciparum has been analyzed and compared with the human host enzyme. The reductive half-reaction is similar to the analogous reaction with GR from other species. The oxidative half-reaction is biphasic, reflecting formation and breakdown of a mixed disulfide between the interchange thiol and GSH. The equilibrium between the E ox-EH2 and GSSG-GSH couples has been modeled showing that the Michaelis complex, mixed disulfide-GSH, is the predominant enzyme form as the oxidative half-reaction progresses; rate constants used in modeling allow calculation of an K eq from the Haldane relationship, 0.075, very similar to the K eq of the same reaction for the yeast enzyme (0.085) (Arscott, L. D., Veine, D. M., and Williams, C. H., Jr. (2000)Biochemistry 39, 4711–4721). Enzyme-monitored turnover indicates that E(FADH−)(S-S)·NADP+ and E(FAD)(SH)2·NADPH are dominant enzyme species in turnover. Since the individual forms of the enzyme differ in their susceptibility to inhibitors, the prevailing states of GR in the cell are of practical relevance. The homodimeric flavoenzyme glutathione reductase (GR) maintains high intracellular concentrations of the antioxidant glutathione (GSSG + NADPH + H+ ↔ 2 GSH + NADP+). Due to its central function in cellular redox metabolism, inhibition of GR from the malarial parasitePlasmodium falciparum represents an important approach to antimalarial drug development; therefore, the catalytic mechanism of GR from P. falciparum has been analyzed and compared with the human host enzyme. The reductive half-reaction is similar to the analogous reaction with GR from other species. The oxidative half-reaction is biphasic, reflecting formation and breakdown of a mixed disulfide between the interchange thiol and GSH. The equilibrium between the E ox-EH2 and GSSG-GSH couples has been modeled showing that the Michaelis complex, mixed disulfide-GSH, is the predominant enzyme form as the oxidative half-reaction progresses; rate constants used in modeling allow calculation of an K eq from the Haldane relationship, 0.075, very similar to the K eq of the same reaction for the yeast enzyme (0.085) (Arscott, L. D., Veine, D. M., and Williams, C. H., Jr. (2000)Biochemistry 39, 4711–4721). Enzyme-monitored turnover indicates that E(FADH−)(S-S)·NADP+ and E(FAD)(SH)2·NADPH are dominant enzyme species in turnover. Since the individual forms of the enzyme differ in their susceptibility to inhibitors, the prevailing states of GR in the cell are of practical relevance. glutathione reductase GR from human placenta 2 electron-reduced GR 4 electron-reduced GR oxidized GR amount of a compound equivalent to 1 FAD-containing subunit glucose 6-phosphate glucose-6-phosphate dehydrogenase mixed disulfide between glutathione reductase and glutathione glutathione reductase fromP. falciparum The glutathione disulfide/glutathione (GSSG/GSH) redox couple is present in most eukaryotic cells at millimolar concentrations (1Jocelyn P.C. Biochemistry of the SH Group. Academic Press, Inc., New York1972: 10-11Google Scholar). Major functions of GSH include the detoxification of reactive oxygen species by donation of reducing equivalents to glutathione peroxidase and glutathione S-transferase as well as enzyme regulation by thiol-disulfide interchange reactions (2Gilbert H.F. Adv. Enzymol. 1990; 63: 69-172PubMed Google Scholar). The flavoenzyme glutathione reductase (GR,1EC 1.6.4.2) catalyzes the reduction of GSSG to GSH and creates an intracellular GSH/GSSG ratio of 20 to 1000 depending on the respective metabolic conditions (3Schirmer R.H. Schulz G.E. Dolphin D. Poulson R. Åvramovic O. Coenzymes and Cofactors, Pyridine Nucleotide Coenzymes: Chemical, Biochemical, and Medical Aspects. John Wiley & Sons, Inc., New York1987: 333-379Google Scholar). Thus, glutathione reductase, a protein of 2 × 55 kDa, plays a key role in cellular redox homeostasis. Both human GR (hGR) and Plasmodium falciparum GR (PfGR) are essential for the survival of the malarial parasite within the human erythrocyte (4Gilberger T.-W. Schirmer R.H. Walter R.D. Müller S. Mol. Biochem. Parasitol. 2000; 107: 169-179Crossref PubMed Scopus (47) Google Scholar, 5Zhang Y. König I. Schirmer R.H. Biochem. Pharmacol. 1988; 37: 861-865Crossref PubMed Scopus (46) Google Scholar). The amino acid sequence identity between the two enzymes is 45% with two major features distinguishing them: (i) an insertion of 34 amino acids within the central domain (residues 318–351) between the FAD binding motif and the highly conserved H8 helix (6Färber P.M. Becker K. Müller S. Schirmer R.H. Franklin R.M. Eur. J. Biochem. 1996; 239: 655-661Crossref PubMed Scopus (54) Google Scholar); (ii) the differences in the amino acid residues lining the wall of the interface cavity, where only 8 out of 25 residues are conserved in PfGR. This cavity represents the binding site of many inhibitors including isoalloxazines, safranine, and menadione in human GR and could be the binding site of methylene blue in PfGR (7Karplus P.A. Pai E.F. Schulz G.E. Eur. J. Biochem. 1989; 178: 693-703Crossref PubMed Scopus (136) Google Scholar). Glutathione reductase belongs to the pyridine nucleotide-disulfide oxidoreductase family of homodimeric flavoenzymes that includes lipoamide dehydrogenase and thioredoxin reductase. Like other members of this family, each subunit of GR contains a disulfide and a flavin that are in redox contact. This ground state of the enzyme is referred to as E ox. In the two-electron-reduced enzyme (EH2) at equilibrium, a rather stable charge transfer complex predominates between one of the nascent thiolates, referred to as the charge transfer or proximal thiolate as the donor, and the flavin, as the acceptor (8Williams Jr., C.H. Müller F. Chemistry and Biochemistry of Flavoenzymes. III. CRC Press, Inc., Boca Raton, FL1992: 121-211Google Scholar). This species can be distinguished from the oxidized (E ox) and fully reduced enzyme (EH4) by its absorbance around 540 nm. The other nascent thiol in EH2 is referred to as the interchange or distal thiol and initiates the dithiol-disulfide interchange with glutathione disulfide that involves a mixed disulfide (MDS) between glutathione and the interchange thiol (7Karplus P.A. Pai E.F. Schulz G.E. Eur. J. Biochem. 1989; 178: 693-703Crossref PubMed Scopus (136) Google Scholar, 9Arscott L.D. Thorpe C. Williams Jr., C.H. Biochemistry. 1981; 20: 1513-1520Crossref PubMed Scopus (85) Google Scholar, 10Pai E.F. Schulz G.E. J. Biol. Chem. 1983; 258: 1752-1757Abstract Full Text PDF PubMed Google Scholar, 11Arscott L.D. Veine D.M. Williams Jr., C.H. Biochemistry. 2000; 39: 4711-4721Crossref PubMed Scopus (36) Google Scholar). Physiologically, E ox is reduced by NADPH via the flavin to give EH2. The several steps of this electron transfer constitute the reductive half-reaction (SchemeFS1). This is followed by an NADP+-NADPH exchange when NADPH is in excess.EH2 then reacts with the second substrate, glutathione disulfide, yielding two molecules of reduced glutathione in the oxidative half-reaction. As previously shown for hGR and the yeast enzyme, a mixed disulfide is an intermediate in the oxidative half-reaction (Scheme FS2) (11Arscott L.D. Veine D.M. Williams Jr., C.H. Biochemistry. 2000; 39: 4711-4721Crossref PubMed Scopus (36) Google Scholar). Evidence for the mixed disulfide was provided by x-ray crystallography using hGR crystals soaked with glutathione (7Karplus P.A. Pai E.F. Schulz G.E. Eur. J. Biochem. 1989; 178: 693-703Crossref PubMed Scopus (136) Google Scholar, 10Pai E.F. Schulz G.E. J. Biol. Chem. 1983; 258: 1752-1757Abstract Full Text PDF PubMed Google Scholar), by chemical identification (12Wong K.K. Vanoni M.A. Blanchard J.S. Biochemistry. 1988; 27: 7091-7096Crossref PubMed Scopus (54) Google Scholar), and by spectral studies (11Arscott L.D. Veine D.M. Williams Jr., C.H. Biochemistry. 2000; 39: 4711-4721Crossref PubMed Scopus (36) Google Scholar). In kinetic studies MDS has been elusive so far (see below).Figure FS3View Large Image Figure ViewerDownload (PPT)Figure 2Reoxidation of P. falciparumglutathione reductase in theEH2(FAD)(SH)2 form by GSSG. A, 21 μm PfGR was reduced with borohydride. Curve 1, 1 eq of GSSG; curve 2, 3 eq; curve 3, 5 eq; curve 4, 10 eq; curve 5, 15 eq; curve 6, 40 eq. The curves were fitted to the sum of 2 exponentials beginning at 5 ms. B, reoxidation of 17 μm EH2 with 1 eq of GSSG in the absence or presence of GSH. The final concentrations of GSH were: curve 1, 0; curve 2, 0.2 mm, curve 3, 0.5 mm; curve 4, 1 mm; curve 5, 2 mm;curve 6, 4 mm; curve 7, 8 mm.View Large Image Figure ViewerDownload (PPT)Figure FS1View Large Image Figure ViewerDownload (PPT)Figure FS2View Large Image Figure ViewerDownload (PPT) Since drug resistance of malarial parasites as well as the geographical distribution of malaria is increasing, new therapeutic approaches are urgently required. Indeed it is clear from the current secular press that global warming, resulting in malaria moving north, has put this disease into the awareness of the “First World.” PfGR represents a potential target of drugs against malaria. The best known lead compound in this context is the antimalarial agent methylene blue, which has been demonstrated to be a specific inhibitor of PfGR (13Färber P.M. Arscott L.D. Williams Jr., C.H. Becker K. Schirmer R.H. FEBS Lett. 1998; 422: 311-314Crossref PubMed Scopus (132) Google Scholar) and does not affect the human enzyme at therapeutic concentrations. The x-ray structure of the methylene blue-PfGR complex has not yet been solved. The most probable binding site for methylene blue is the intersubunit cavity of the enzyme. The hGR, on the other hand, is arguably the most thoroughly studied flavoenzyme by x-ray crystal methods (7Karplus P.A. Pai E.F. Schulz G.E. Eur. J. Biochem. 1989; 178: 693-703Crossref PubMed Scopus (136) Google Scholar). To develop parasite-specific drugs it is essential to understand and compare kinetic, functional, and structural properties of parasite and host cell enzymes and to identify the enzyme species that predominatein vivo, therefore serving as antiparasitic drug targets. In the present study we have investigated the reductive and oxidative half-reactions of PfGR. With the aid of computer simulation programs, we offer the first kinetic evidence for the MDS form. Furthermore, we studied the protein in enzyme-monitored turnover and characterized enzyme intermediates that can be considered as targets of syncatalytic inhibitors. Some of these intermediates such asEH2·NADPH and the newly identified MDS are likely to occur in the cytosol of parasites that are not challenged by reactive oxygen species. Under these conditions, most cytosolic GR molecules are at equilibrium. EH4, containing both the flavin and the active site cysteine pair in the reduced form, was identified by reducing PfGR with NADPH under forcing conditions. Since the individual forms of the enzyme differ in their susceptibility to inhibitors, the characterization of these newly identified species is of high practical relevance. Recombinant PfGR was expressed inEscherichia coli SG5 cells and purified by affinity chromatography on 2′,5′-ADP-Sepharose as described previously (13Färber P.M. Arscott L.D. Williams Jr., C.H. Becker K. Schirmer R.H. FEBS Lett. 1998; 422: 311-314Crossref PubMed Scopus (132) Google Scholar). GSH, GSSG, NADPH, Leuconostoc mesenteroidesglucose-6-phosphate dehydrogenase, glucose 6-phosphate, methylene blue, dithionite, and all other reagents were obtained from Serva and Sigma. All reagents used were of the highest purity available. The rapid reaction kinetics of the enzyme were measured under anaerobic conditions in a stopped flow spectrophotometer as described before (14Rietveld P. Arscott L.D. Berry A. Scrutton N.S. Deonarain M.P. Perham R.N. Williams Jr., C.H. Biochemistry. 1994; 33: 13888-13895Crossref PubMed Scopus (74) Google Scholar). Solutions were made anaerobic using alternating cycles of vacuum and nitrogen (15Williams Jr., C.H. Arscott L.D. Matthews R.G. Thorpe C. Wilkinson K.D. McCormick D.B. Wright L.D. Methods in Enzymology: Vitamins and Coenzymes. Academic Press, Inc., New York1979: 185-198Google Scholar). All reactions were performed in GR buffer containing 47 mmpotassium phosphate, 200 mm KCl, and 1 mm EDTA, pH 6.9, at 4 °C. After mixing, the PfGR concentration varied between 15 and 22 μm. The data were analyzed by curve-fitting either to a single or to multiple exponential functions (16Marquardt D.W. J. Mol. Biol. 1963; 95: 701-729Google Scholar) in a program written by D. A. Ballou, University of Michigan. To determine the maximal rate constants under saturating conditions and the apparent K d, rate constants (k obs) as a function of the substrate concentration were fitted to a rectangular hyperbola. PfGR was reduced by a 40-fold excess sodium borohydride over E ox. The NaBH4 had been dissolved in 20 μl of 0.2 m NaOH. AfterEH2 was fully formed and excess borohydride hydrolyzed at a rate of 0.1 s−1, pH 6.9, oxidation was started by mixing with GSSG (17Davis R.E. Swain C.G. J. Am. Chem. Soc. 1960; 82: 5949-5950Crossref Scopus (71) Google Scholar). The free acid form of GSH used in these experiments was dissolved in anaerobic GR buffer and then titrated to pH 6.9 using 1 m NaOH. Quantitation of the thiol titer was determined using 5,5′-dithiobis(2-nitrobenzoic acid). The content of GSSG in the GSH stock solutions varied from batch to batch between 0.5 and 1%. To anaerobic solutions of 1 eq of GSSG, varied amounts of anaerobic GSH were added. These GSSG/GSH solutions were mixed rapidly with the reduced PfGR, and the spectral changes were followed in the stopped flow for all regions of interest. For experiments where constant NADPH concentrations were required, an NADPH-regenerating system consisting of glucose-6-phosphate dehydrogenase (G6PDH) and glucose 6-phosphate (G6P) was employed. For the titration experiment, the anaerobic cuvette contained 5 mm G6P and 5 units/ml G6PDH (at 25 °C), the NADPH concentration in the side arm being 25 μm. For stopped flow experiments the G6PDH activity was raised to 1000 units/ml. The spectral properties of the enzyme species detected with PfGR are summarized in Table I, which is patterned after a similar table in Krauth-Siegel et al. (18Krauth-Siegel R.L. Arscott L.D. Schönleben-Janas A. Schirmer R.H. Williams Jr., C.H. Biochemistry. 1998; 37: 13968-13977Crossref PubMed Scopus (59) Google Scholar). During the reductive half-reaction with excess NADPH, E ox is reduced to EH2(FAD)(SH)2·NADPH via the four intermediates shown (Table I, top to bottom).EH2 is thus a mixture of several forms; the predominant species is the thiolate-flavin charge transfer complex,EH2(FAD)(SH)2.EH2(FAD)(SH)2 forms complexes with NADPH and NADP+. The spectral characteristics of the charge transfer complexes and the four-electron-reduced enzymeEH4 at pH 6.9 were determined in the experiments described below and are essential for the interpretation of the pre-steady state kinetic results.Table ISpectral properties of various PfGR enzyme speciesEnzyme speciesCharacteristic wavelengthε at this wavelengthε at 450 nmnmmm −1 cm −1mm −1 cm −1E ox46011.310E ox·NADPH5709.8EH2(FADH−)(S-S)·NADP+680–7202.1 (720 nm)2.6EH2(FAD)(SH)25402.89.9EH2(FAD)(SH)2·NADP+6008.4EH2(FAD)(SH)2·NADPH5404.48.2EH44502.52.5 Open table in a new tab Anaerobic titration of PfGR with dithionite requires 1 eq/FAD-containing subunit to giveEH2(FAD)(SH)2 and a second equivalent to form EH4 (data not shown). As expected, there were no other redox active centers in PfGR. Titration of E ox with the physiological reductant NADPH leads to theEH2(FAD)(SH)2·NADP+charge transfer complex with a K d of less than 3 μm; excess NADPH displaced the NADP+ (data not shown). Rapid reaction kinetics for the reductive half-reaction with NADPH revealed four distinct phases that could be detected at 440, 540, and 670 nm. The two faster steps were dependent on the NADPH concentration, the first being clearly attributable to FAD reduction (14Rietveld P. Arscott L.D. Berry A. Scrutton N.S. Deonarain M.P. Perham R.N. Williams Jr., C.H. Biochemistry. 1994; 33: 13888-13895Crossref PubMed Scopus (74) Google Scholar). Fig.1 shows the spectra recorded during the reaction of the enzyme with 4 eq of NADPH. The first phase (spectra 1–3) is characterized by a decrease at 460 nm and an increase at wavelengths greater than 600 nm, which indicates formation of theEH2(FADH−)(S-S)·NADP+complex. TheEH2(FADH−)(S-S)·NADP+species is formed and partially degraded too rapidly for the full extent of its formation to be observed (14Rietveld P. Arscott L.D. Berry A. Scrutton N.S. Deonarain M.P. Perham R.N. Williams Jr., C.H. Biochemistry. 1994; 33: 13888-13895Crossref PubMed Scopus (74) Google Scholar). The phase giving rise to spectra 1–3 is easily distinguished from a slower kinetic process (spectra 4–6), at the end of which the spectrum is typical of theEH2(FAD)(SH)2·NADPH complex. This is reinforced by the presence of an isosbestic point at 620 nm, indicating that from 17 to 300 ms one enzyme species is being converted to another. The four phases were well resolved in the kinetic trace shown in the inset of Fig. 1; the rate constants are given in Table II and shown to be comparable with those measured with the E. coli enzyme. The fourth phase, having a rate of 4 s−1, was associated with a very small fraction of the total absorbance change; it may have been due to ongoing exchange of NADP+ by the excess of NADPH.Table IIComparison of rate constants of glutathione reductase from different sourcesk appPfGRhGR 2-aRef. 18.E. coli GR 2-bRef. 14.Yeast GR 2-cRef. 41 and (L. D. Arscott and C. H. Williams, unpublished data).s −1s −1s −1s −1Reductive half-reaction k 1350500330153 k 2290270 k 34510011068 k 44614Oxidative half-reaction k 12364901200 k 25445–512-a Ref. 18Krauth-Siegel R.L. Arscott L.D. Schönleben-Janas A. Schirmer R.H. Williams Jr., C.H. Biochemistry. 1998; 37: 13968-13977Crossref PubMed Scopus (59) Google Scholar.2-b Ref. 14Rietveld P. Arscott L.D. Berry A. Scrutton N.S. Deonarain M.P. Perham R.N. Williams Jr., C.H. Biochemistry. 1994; 33: 13888-13895Crossref PubMed Scopus (74) Google Scholar.2-c Ref. 41Huber P.W. Brandt K.G. Biochemistry. 1980; 19: 4568-4575Crossref Scopus (30) Google Scholar and (L. D. Arscott and C. H. Williams, unpublished data). Open table in a new tab Reduction of PfGR with borohydride led to the thiolate-flavin charge transfer complex,EH2(FAD)(SH)2, with its characteristic absorbance at 540 nm. During the subsequent oxidation with glutathione disulfide the absorbance at 540 nm decreased continuously, which is shown in the kinetic traces in Fig.2 A. The reaction appeared to be biphasic, and both rates were dependent on the GSSG concentration; the first phase was associated with up to 90% of the absorbance change and had a limiting rate of 236 s−1(K d,app = 346 μm); the second phase had a limiting rate of 54 s−1(K d,app = 225 μm). Chemical precedent shows that the reaction is composed of two steps, formation and breakdown of a MDS between glutathione and the interchange thiol of GR (11Arscott L.D. Veine D.M. Williams Jr., C.H. Biochemistry. 2000; 39: 4711-4721Crossref PubMed Scopus (36) Google Scholar). However, the observed rates reflect combinations of rate constants and are not directly attributable to individual reactions (see modeling below). The fitted curves in Fig.2 A all tend to the same starting absorbance; the real data, starting at approximately 5 ms, began at continuously lower values as the GSSG concentration was raised. Since the formation of MDS involves only a small extinction change, this indicates that most of the MDS formation has taken place in the dead time; this is born out by the model (below). Thus, most of the observed reaction describes the breakdown of the MDS. PfGR is the only verified enzymic target of the antimalarial dye methylene blue (13Färber P.M. Arscott L.D. Williams Jr., C.H. Becker K. Schirmer R.H. FEBS Lett. 1998; 422: 311-314Crossref PubMed Scopus (132) Google Scholar, 19Guttmann P. Ehrlich P. Berl. Klin. Wochenschr. 1891; 28: 953-956Google Scholar, 20Vennerstrom J.L. Makler M.T. Angerhofer C.K. Williams J.A. Antimicrob. Agents Chemother. 1995; 39: 2671-2677Crossref PubMed Scopus (194) Google Scholar). The addition of 1–3 eq of inhibitor in the oxidative half-reaction did not affect the kinetic characteristics of the reaction. The reductive half-reaction, however, appeared to be altered in the presence of the inhibitor, but priorities for the limited enzyme precluded further study of the effect. The mixed disulfide had been predicted in earlier work (9Arscott L.D. Thorpe C. Williams Jr., C.H. Biochemistry. 1981; 20: 1513-1520Crossref PubMed Scopus (85) Google Scholar). Direct evidence for this species was provided by x-ray crystallography using GR crystals soaked with glutathione (7Karplus P.A. Pai E.F. Schulz G.E. Eur. J. Biochem. 1989; 178: 693-703Crossref PubMed Scopus (136) Google Scholar, 10Pai E.F. Schulz G.E. J. Biol. Chem. 1983; 258: 1752-1757Abstract Full Text PDF PubMed Google Scholar) and by chemical modifications (12Wong K.K. Vanoni M.A. Blanchard J.S. Biochemistry. 1988; 27: 7091-7096Crossref PubMed Scopus (54) Google Scholar). The MDS was assumed to provide a promising target for rational drug design (7Karplus P.A. Pai E.F. Schulz G.E. Eur. J. Biochem. 1989; 178: 693-703Crossref PubMed Scopus (136) Google Scholar). The spectral characteristics of the MDS of yeast GR have recently been described (11Arscott L.D. Veine D.M. Williams Jr., C.H. Biochemistry. 2000; 39: 4711-4721Crossref PubMed Scopus (36) Google Scholar). Evidence for the MDS between the interchange thiol of the enzyme (Cys-58) and glutathione could be observed readily when GSH is included together with the GSSG in the oxidative half-reaction.EH2 was no longer quantitatively oxidized by GSSG to E ox as in the absence of GSH, because an intermediate, attributed to the mixed disulfide, accumulates. The kinetic traces observed at several concentrations of GSH are shown in Fig. 2 B and compared with the kinetics observed in the absence of GSH. The MDS was found to have a lower thiolate-flavin charge transfer absorbance and a higher 462 nm/492 nm ratio than seen in EH2(FAD)(SH)2. As more GSH was added to the oxidative half-reaction, breakdown of the MDS was retarded, and more MDS accumulated. Fig.3 shows the change in the 462 nm/492 nm ratio as the reaction ofEH2(FAD)(SH)2 (ratio of approximately 1.34) with GSSG progressed to the MDS (ratio of approximately 1.42) in the presence of GSH and toE ox (ratio of approximately 1.26) in the absence of GSH. Analysis of the spectra of the enzyme species formed during the oxidative half-reaction by singular value decomposition revealed three eigenvectors when GSH was included in the reaction mixture, whereas in the absence of GSH, only two significant eigenvectors were observed, representing E ox andEH2(FAD)(SH)2; the third eigenvector that developed in the presence of GSH was attributed to the MDS. Since the formation of MDS fromEH2(FAD)(SH)2 involves a small absorbance change and MDS breakdown to E oxinvolves a large absorbance change, the isosbestics at 355, 390, 450, and 508 nm, observed for interconversion of E oxand EH2(FAD)(SH)2, are not expected in the spectra of the MDS approximated by singular value decomposition. The approximations are given in Fig. 4for three concentrations of GSH. A model for the kinetic equilibrium between theE ox-EH2 and the GSSG-GSH redox couples is presented in Scheme FS3. The reaction has been simulated in the direction ofEH2(FAD)(SH)2 oxidation by GSSG using the set of rate constants shown in TableIII; the extinction coefficients at 540 nm of the enzyme forms were held constant in the simulations:EH2(FAD)(SH)2 = 3000; MDS = 2650; E ox = 535m−1 cm−1. The quality of the simulation is shown in Fig.5 A and can be compared with the fitted curves shown in Fig. 2 A. Fig. 5 B shows the mole fractions of the six species as the reaction ofEH2(FAD)(SH)2 with 40 eq of GSSG progresses. It can be seen that MDS·GSH is the predominant intermediate; it represents 48% at 3.4 ms; the reaction reaches equilibrium at approximately 50 ms and is essentially complete with 95% E ox and no detectableEH2(FAD)(SH)2. IfEH2(FAD)(SH)2 is reoxidized by stoichiometric GSSG in the presence of excess GSH, MDS·GSH remains the predominate intermediate, 46% at 250 ms when the reaction has reached equilibrium with 4% E ox and 33%EH2 (data not shown). The results are predicted by the model since MDS·GSH precedes the rate-limiting step in which the first molecule of GSH dissociates with a rate constant,k 5 = 250 s−1. The simulations of the experiments with added GSH (Fig. 5 A,curves 7 and 8) are not as good as the others (curves 1–6). The limiting observed rate of 236 s−1 is associated with up to 90% of theA 540 nm disappearance and probably reflects the breakdown of the MDS limited by the dissociation of the first molecule of GSH (Table II). The limiting observed rate of 54 s−1, involving as little as 10% of theA 540 nm disappearance, is not easily associated with a chemical step in the model. Fitting theA 540 nm to 2 exponentials assumes a simple A to B to C model and should not be compared with the more complete A to C to D to E to G to H model of Scheme FS3.Table IIIModeling of equilibria important in the oxidative half-reaction according to Scheme FS3ParameterSimulation rate constantsMeasured rate constantsMeasuredK d appSimulation K dk 1 (m−1s−1)1.5 × 106NM 3-aNM, not measured.346 μm346 μmk 2 (s−1)520NMk 3 (s−1)800NMk 4 (s−1)12 3k 5 (s−1)250236k 6 (m−1s−1)1 × 106NM225 μm250 μmk 7(s−1)440311 3-bData from yeast GR (L. D. Arscott and C. H. Williams, unpublished).k 8(s−1)280185 3-bData from yeast GR (L. D. Arscott and C. H. Williams, unpublished).k 9 (s−1)1200NMk 10 (m−1s−1)1.2 × 106NM0.35 to 2 mm 3-bData from yeast GR (L. D. Arscott and C. H. Williams, unpublished).1 mm3-a NM, not measured.3-b Data from yeast GR (L. D. Arscott and C. H. Williams, unpublished). Open table in a new tab The reaction has also been simulated in the opposite direction using data from the reduction of E ox by GSH and the rate constants in Table III. The quality of the simulation is shown in Fig. 5 C. GSH dissociation from MDS is set at 250 s−1 in both simulations, and it can be argued that there is no reason that the dissociation of GSH from MDS (k 5) or from E ox(k 9) should be the same because different binding sites (1GSH and 2GSH) are involved (10Pai E.F. Schulz G.E. J. Biol. Chem. 1983; 258: 1752-1757Abstract Full Text PDF PubMed Google Scholar). Fig. 5 D shows the mole fractions of the six species as the reaction of E ox with 100 eq of GSH progresses. It can be seen that MDS·GSH is the major intermediate; MDS·GSH and the product,EH2(FAD)(SH)2, are both present at approximately 35% at 1 s, when the reaction has reached equilibrium. Again, the model predicts the buildup of MDS·GSH, since this intermediate precedes the rate-limiting step,k 4, of 12 s−1. To determine the predominant PfGR species under turnover conditions in the cell, PfGR was monitored during in vitro turnover under quasi-physiological conditions. The kinetic traces at 460 nm for three experiments are shown in Fig. 6 A: NADPH with excess GSSG in the absence of GSH (curve 1), NADPH, and GSSG at equal concentrations in the absence and presence of GSH (curves 2 and 3). Three major phases can be distinguished during the reaction: PfGR reduction with NADPH, steady state turnover, and reoxidation by GSSG. After PfGR reduction, turnover continued until NADPH was used up by GSSG. The spectrum acquired just before turnover is spectrum 2 in Fig. 6 B. It seems to represent a mixture of two enzyme species, namely (i)EH2(FADH−)(S-S)·NADP+, which is indicated by the long wavelength band at 680 nm, and (ii) theEH2(FAD)(SH)2·NADPH complex as indicated by the increased absorbance due to the thiolate-flavin charge transfer complex at 540 nm (Table I). These two enzyme species are involved in the rate-limiting step of the reductive half-reaction, namely the transfer of reducing equivalents from FAD to form the dithiol. As the reaction progresses into turnover, the latter enzyme species becomes predominant (spectrum 3 in the presence of GSH and spectrum 4 in its absence). The high NADPH concentration in many cells is due to the action of NADPH-regenerating enzymes such as glucose-6-phosphate dehydrogenase. The G6PDH/G6P system was used in a static experiment to reduce PfGR. In such an experiment, the concentration of NADP+ was vanishingly low as it was constantly reduced to NADPH by G6PDH, resulting in a high ratio of NADPH/NADP+. In contrast to a simple titration with NADPH, E ox is not only reduced to EH2 but slowly toEH4, the fully reduced species of PfGR (Fig.6 B, curve 7), suggesting thatEH4 could be formed under cellular conditions (see below). Spectral characterist" @default.
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W2079548047 title "Kinetic Characterization of Glutathione Reductase from the Malarial Parasite Plasmodium falciparum" @default.
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