Role of Active Site Tyrosine Residues in Catalysis by Human Glutathione Reductase

Rl, Krauth-Siegel; Ld, Arscott; Schönleben-Janas, A.; Rh, Schirmer; Ch, Williams

doi:10.1021/bi980637j

Cited by 73 publications

(77 citation statements)

References 30 publications

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“…Although NaBH 4 is a strong reductant, it does not reduce DmTrxR-1 to EH 6 , but rather to a reduced enzyme species with strong CT absorption (⑀ 540 nm ϭ 3.3 mM Ϫ1 cm Ϫ1 ) that is similar to the EH 4 spectrum obtained from reduction with NADPH (⑀ 540 nm ϳ 4.0 mM Ϫ1 cm Ϫ1 ). The extinctions are not the same because NADPH enhances the CT (22). Four eq of ferricyanide were required to restore the E ox spectrum, which is consistent with the assumption that borohydride produced the EH 4 form of TrxR and that two reducible disulfides occur in the enzyme.…”

Section: Resultssupporting

confidence: 69%

“…3, left panel). Hydride transfer from the first NADPH to the flavin is observed as the formation, at a rate of ϳ95-120 s Ϫ1 (within the first 30 ms), of a broad spectral band centered at 680 nm; in this charge-transfer complex, reduced flavin is the donor and NADP ϩ is the acceptor (22). With some flavoprotein disulfide reductases, this cannot be observed because the subsequent formation of the thiolate-flavin charge-transfer species is too fast.…”

Section: Discussionmentioning

confidence: 99%

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The Mechanism of High M r Thioredoxin Reductase from Drosophila melanogaster

Bauer

Massey

Arscott

et al. 2003

Journal of Biological Chemistry

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Drosophila melanogaster thioredoxin reductase-1 (DmTrxR-1) is a key flavoenzyme in dipteran insects, where it substitutes for glutathione reductase. DmTrxR-1 belongs to the family of dimeric, high M r thioredoxin reductases, which catalyze reduction of thioredoxin by NADPH. Thioredoxin reductase has an N-terminal redox-active disulfide (Cys 57 -Cys 62 ) adjacent to the flavin and a redox-active C-terminal cysteine pair (Cys 489 The first equivalent yields a FADH ؊ -NADP ؉ chargetransfer complex that reduces the adjacent disulfide to form a thiolate-flavin charge-transfer complex. EH 4 reacts with thioredoxin rapidly to produce EH 2 . In contrast, E ox formation is slow and incomplete; thus, EH 2 of wild-type cannot reduce thioredoxin at catalytically competent rates. Mutants lacking the C-terminal redox center, C489S, C490S, and C489S/C490S, are incapable of reducing thioredoxin and can only be reduced to EH 2 forms. Additional data suggest that Cys 57 attacks Cys 490 in the interchange reaction between the N-terminal dithiol and the C-terminal disulfide.Thioredoxin reductase (TrxR) 1 catalyzes the NADPHdependent reduction of the redox-active disulfide of thioredoxin (Trx), a 12-kDa protein. The thioredoxin system is widely distributed in nature, and in most organisms it functions in concert with the glutathione system. However, because insects such as Drosophila melanogaster have no glutathione reductase, glutathione disulfide is reduced non-enzymatically by reduced Trx (1); thus, TrxR serves an additional role in insects. TrxR from higher eukaryotes, including D. melanogaster, is of the high molecular weight type, having a M r of 54,000 -58,000, which contrasts with the well studied TrxR of Escherichia coli with a M r of 34,000. There is an interesting difference in the mechanism whereby these enzymes transfer reducing equivalents from the protein interior to the enzyme surface where Trx is bound and reduced. In low M r TrxR, one domain rotates from a conformation in which the redox-active disulfide is reduced by the flavin in the apolar interior to a conformation in which the nascent dithiol is carried to the hydrophilic surface of the enzyme, where it can reduce bound Trx. In this unusual mechanism, when the dithiol is near the surface to reduce Trx, the NADPH is in position to reduce the FAD (2). High M r thioredoxin reductases, on the other hand, have a second redox-active disulfide or selenosulfide pair that shuttles reducing equivalents from the nascent dithiol that is near the flavin to Trx bound at the surface (3).High M r TrxRs are part of the disulfide reductase family that includes lipoamide dehydrogenase, glutathione reductase, mercuric reductase, trypanothione reductase, and peroxiredoxin reductase. All of these flavoenzymes are homodimers (4), and each monomer comprises three domains: a flavin binding domain, a pyridine nucleotide binding domain, and a domain that provides an interface between the two monomers, as shown in Scheme 1. Each active site of thioredoxin reductase contains FAD and a s...

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Section: Resultssupporting

confidence: 69%

Section: Discussionmentioning

confidence: 99%

The Mechanism of High M r Thioredoxin Reductase from Drosophila melanogaster

Bauer

Massey

Arscott

et al. 2003

Journal of Biological Chemistry

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“…Characteristics of PfGR-The spectral properties of the enzyme species detected with PfGR are summarized in Table I, which is patterned after a similar table in KrauthSiegel et al (18). During the reductive half-reaction with excess NADPH, E ox is reduced to EH 2 (FAD)(SH) 2 ⅐NADPH via the four intermediates shown (Table I, top to bottom).…”

Section: Spectralmentioning

confidence: 81%

Kinetic Characterization of Glutathione Reductase from the Malarial Parasite Plasmodium falciparum

Böhme¹,

Arscott²,

Becker³

et al. 2000

Journal of Biological Chemistry

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The homodimeric flavoenzyme glutathione reductase (GR) maintains high intracellular concentrations of the antioxidant glutathione (GSSG ؉ NADPH ؉ H ؉ 7 2 GSH ؉ NADP ؉ ). Due to its central function in cellular redox metabolism, inhibition of GR from the malarial parasite Plasmodium 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 halfreaction is biphasic, reflecting formation and breakdown of a mixed disulfide between the interchange thiol and GSH. The equilibrium between the E ox -EH 2 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 ( (S-S)⅐NADP؉ and E(FAD)(SH) 2 ⅐NADPH 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 glutathione disulfide/glutathione (GSSG/GSH) redox couple is present in most eukaryotic cells at millimolar concentrations (1). 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 (2). The flavoenzyme glutathione reductase (GR, 1 EC 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 (3). Thus, glutathione reductase, a protein of 2 ϫ 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 (4, 5). 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 (6); (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 (7).Glutathione reductase belongs to the pyridine nucleotidedisulfide 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 ...

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“…Subsequent auto-oxidation led to partially reoxidized protein-bound flavin and indirectly to the production of NADP ϩ (gray curve). Here, the characteristic broad peak at around 680 nm represents the complex between protein-bound FADH Ϫ and NADP ϩ (33). The original spectrum of oxidized proteinbound flavin (black curve) was recovered after 20 min by auto-oxidation and was 10 times faster by oxidation with 3 M MB (Fig.…”

Section: Discussionmentioning

confidence: 97%

Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues fromPlasmodium falciparum

Buchholz

Schirmer

Eubel

et al. 2008

Antimicrob Agents Chemother

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Methylene blue (MB) has experienced a renaissance mainly as a component of drug combinations against Plasmodium falciparum malaria. Here, we report biochemically relevant pharmacological data on MB such as rate constants for the uncatalyzed reaction of MB at pH 7.4 with cellular reductants like NAD(P)H (k ‫؍‬ 4 M ؊1 s ؊1 ), thioredoxins (k ‫؍‬ 8.5 to 26 M ؊1 s ؊1 ), dihydrolipoamide (k ‫؍‬ 53 M ؊1 s ؊1 ), and slowly reacting glutathione. As the disulfide reductases are prominent targets of MB, optical tests for enzymes reducing MB at the expense of NAD(P)H under aerobic conditions were developed. The product leucomethylene blue (leucoMB) is auto-oxidized back to MB at pH 7 but can be stabilized by enzymes at pH 5.0, which makes this colorless compound an interesting drug candidate. MB was found to be an inhibitor and/or a redox-cycling substrate of mammalian and P. falciparum disulfide reductases, with the k cat values ranging from 0.03 s ؊1 to 10 s ؊1 at 25°C. Kinetic spectroscopy of mutagenized glutathione reductase indicates that MB reduction is conducted by enzyme-bound reduced flavin rather than by the active-site dithiol Cys 58 /Cys 63 . The enzyme-catalyzed reduction of MB and subsequent auto-oxidation of the product leucoMB mean that MB is a redox-cycling agent which produces H 2 O 2 at the expense of O 2 and of NAD(P)H in each cycle, turning the antioxidant disulfide reductases into pro-oxidant enzymes. This explains the terms subversive substrate or turncoat inhibitor for MB. The results are discussed in cellpathological and clinical contexts. Methylene blue (MB or MBϩ ) [also known as methylthionine hydrochloride or 3,7-bis(dimethylamino)phenothiazin-5-ium chloride] was the very first synthetic compound to be used as a drug. Paul Ehrlich, who introduced the concept of modern target-based chemotherapy using MB as an example, and Paul Guttmann described the compound as being an effective antimalarial agent (28). Despite its beneficial antimalarial activity, the drug disappeared from the scene because up-and-coming compounds such as chloroquine were more effective; in addition, soldiers resented taking MB because of inevitable but harmless side effects: green or blue urine and bluish sclerae (47, 55).After MB was revisited as an antimalarial agent (6, 53) and found to be an inhibitor of Plasmodium falciparum glutathione (GSH) reductase (GR) (23), it was studied as a partner drug in antimalarial drug combinations (2,39,48,51).Compared with other antiparasitic agents, MB is affordable and registered in most countries (as the treatment of choice for acute and chronic methemoglobinemia [16,17,36]), and it can be made internationally available in sufficient dosages (51). The price for treating a malaria episode in a child with an MB-containing drug combination would be less than €0.50 (40). Drug resistance has not been reported for MB and could not be provoked in rodent malaria models (52, 53).Because of its favorable properties, including the differential staining of cell biological structures and protein cr...

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Role of Active Site Tyrosine Residues in Catalysis by Human Glutathione Reductase

Cited by 73 publications

References 30 publications

The Mechanism of High M r Thioredoxin Reductase from Drosophila melanogaster

The Mechanism of High M r Thioredoxin Reductase from Drosophila melanogaster

Kinetic Characterization of Glutathione Reductase from the Malarial Parasite Plasmodium falciparum

Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues fromPlasmodium falciparum

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