The binding of the retro-analogue of glutathione disulfide to glutathione reductase.

Janes, Wolfgang; Schulz, Georg E.

doi:10.1016/s0021-9258(18)86966-6

Cited by 16 publications

(43 citation statements)

References 9 publications

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“…The main location coincides with that in E and EH 2 (which differ by only about 0.1 Å) as well as several enzyme species with covalently modified Cys58. 16,17,[21][22][23] A refinement cycle including both conformations for Cys63 resulted in a model that fits well the (F obs -F calc ) exp iR calc and (2F obs -F calc ) exp iR calc electron density maps (Figure 3). In the alternate conformation Cys63 does not seem to form a disulfide bridge with Cys58 since the distance of 5.5 Å is too long and negative difference density appeared between Cys58 and Cys63 if the disulfide bridge was built with Cys63 in this conformation.…”

Section: Inhibition Of Human Gr and T Cruzi Tr Bymentioning

confidence: 81%

“…The additional electron density for Cys58 corresponds to the position of the residue in the oxidized enzyme and reveals that some of the protein is not modified but forms the disulfide bridge with Cys63 (coarse dashed line). The major conformation of Cys63 is that usually observed in the oxidized and reduced enzyme as well as in several protein species modified at Cys58. ,,− In the ajoene-modified GR, Cys63 shows an alternative conformation (marked by an asterisk; for details see text). The contour level is 2.1σ.…”

Section: Resultsmentioning

confidence: 99%

“…The major conformation of Cys63 is that usually observed in the oxidized and reduced enzyme as well as in several protein species modified at Cys58. 16,17,[21][22][23] In the ajoene-modified GR, Cys63 shows an alternative conformation (marked by an asterisk; for details see text). The contour level is 2.1σ.…”

Section: Inhibition Of Human Gr and T Cruzi Tr Bymentioning

confidence: 99%

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Ajoene Is an Inhibitor and Subversive Substrate of Human Glutathione Reductase and Trypanosoma cruzi Trypanothione Reductase: Crystallographic, Kinetic, and Spectroscopic Studies

et al. 1999

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Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a garlic-derived natural compound, is a covalent inhibitor as well as a substrate of human glutathione reductase (GR) and Trypanosoma cruzi trypanothione reductase (TR). The 2.1-A resolution crystal structure of GR inhibited by (E)-ajoene revealed a mixed disulfide between the active site Cys58 and the CH2=CH-CH2-SO-CH2-CH=CH-S moiety of ajoene. The modified enzyme has a markedly increased oxidase activity when compared to free GR. GR reduces (Z)-ajoene with a kcat/Km of 6.8 x 10(3) M-1 s-1 yielding 4,5,9-trithiadodeca-1, 6,11-triene (deoxyajoene) and 4,8,9,13-tetrathiahexadeca-1,6,10, 15-tetraene as stable reaction products. The reaction leads also to the formation of single-electron reduced products and concomitantly superoxide anion radicals as shown by coupling the reaction to the reduction of cytochrome c. The interactions between the flavoenzymes and ajoene are expected to increase the oxidative stress of the respective cell. The antiparasitic and cytostatic actions of ajoene may at least in part be due to the multiple effects on key enzymes of the antioxidant thiol metabolism.

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Section: Inhibition Of Human Gr and T Cruzi Tr Bymentioning

confidence: 81%

Section: Resultsmentioning

confidence: 99%

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Ajoene Is an Inhibitor and Subversive Substrate of Human Glutathione Reductase and Trypanosoma cruzi Trypanothione Reductase: Crystallographic, Kinetic, and Spectroscopic Studies

et al. 1999

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“…The structures of GRs from Escherichia coli (Mittl & Schulz, 1994) and from human red blood cells have been determined (Schulz et al, 1978); that of the human enzyme has been solved to a resolution of 1.54 Å (Karplus & Schulz, 1987). Extensive analyses of its complexes with its natural substrate GSSG and with a variety of different glutathione analogues have been described Janes & Schulz, 1990;. Crystal structures of TRs from Crithidia fasciculata (Kuriyan et al, 1991;Hunter et al, 1992) and from Trypanosoma cruzi have been solved (Lantwin et al, 1994).…”

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confidence: 99%

Glutathione Reductase Turned into Trypanothione Reductase: Structural Analysis of an Engineered Change in Substrate Specificity^,

et al. 1997

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Trypanosoma and Leishmania, pathogens responsible for diseases such as African sleeping sickness, Chagas' heart disease, or Oriental sore, are two of the very few genera that do not use the ubiquitous glutathione/glutathione reductase system to keep a stable cellular redox balance. Instead, they rely on trypanothione and trypanothione reductase to protect them from oxidative stress. Trypanothione reductase (TR) and the corresponding host enzyme, human red blood cell glutathione reductase (GR), belong to the same flavoprotein family. Despite their closely related three-dimensional structures and although their natural substrates share the common structural glutathione core, the two enzymes are mutually exclusive with respect to their disulfide substrates. This makes the parasite enzyme a potential target for antitrypanosomal drug design. While a large body of structural data on GR complexes is available, information on TR-ligand interactions is very limited. When the two amino acid changes Ala34Glu and Arg37Trp are introduced into human GR, the resulting mutant enzyme (GRTR) prefers trypanothione 700-fold over its original substrate, effectively converting a GR into a TR [Bradley, M., Bücheler, U. S.,& Walsh, C. T. (1991) Biochemistry 30, 6124-6127]. The crystal structure of GRTR has been determined at 2.3 A resolution and refined to a crystallographic R factor of 20.9%. We have taken advantage of the ease with which ligand complexes can be produced in GR crystals, a property that extends to the isomorphous GRTR crystals, and have produced and analyzed crystals of GRTR complexes with glutathione, trypanothione, glutathionylspermidine and of a true catalytic intermediate, the mixed disulfide between trypanothione and the enzyme. The corresponding molecular structures have been characterized at resolutions between 2.3 and 2.8 A with R factors ranging from 17.1 to 19.7%. The results indicate that the Ala34Glu mutation causes steric hindrance leading to a large displacement of the side chain of Arg347. This movement combined with the change in charge introduced by the mutations modifies the binding cavity, forcing glutathione to adopt a nonproductive binding mode and permitting trypanothione and to a certain degree also the weak substrate glutathionylspermidine to assume a productive mode.

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“…14 The crystal structure of an end group-modified retro-inVerso isomer of glutathione disulfide bound to glutathione reductase also was obtained to explain why it is a poor substrate for the enzyme. 15 Electron-nuclear double resonance (ENDOR) spectroscopy has proven to be a sensitive tool for determining the structure of paramagnetic centers in enzyme active sites. [16][17][18] We recently showed that 35 GHz Mims pulsed ENDOR spectroscopy is surprisingly effective for studying non-covalently bound substrates in the vicinity of the high spin ferriheme of fully functional holo-NOSs, through measurements that determined the binding geometry of L-arginine (L-Arg), 19,20 and both the binding geometry and protonation state of N G -hydroxy-Larginine (NOHA).…”

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confidence: 99%

ENDOR Spectroscopic Evidence for the Geometry of Binding of retro-inverso-N^ω-Nitroarginine-Containing Dipeptide Amides to Neuronal Nitric Oxide Synthase

Tierney¹,

Huang²,

Martásek

et al. 2000

J. Am. Chem. Soc.

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We describe in detail the use of 35 GHz Mims pulsed 15N and 1,2H electron−nuclear double resonance (ENDOR) spectroscopy to study the binding of substrates and inhibitors to nitric oxide synthase (NOS). We show that reliable distance estimates, and limited orientation information, can be derived from a small set of data taken near the peak of the absorption mode EPR signal, while more precise orientations require a more extensive data set. The ENDOR approach is then applied to the binding of isoform-selective and non-selective nitroarginine inhibitors. Recently, we reported a family of N ω-nitroarginine-containing dipeptide amides as highly selective inhibitors of nNOS (Huang, H. et al. J. Med. Chem. 1999, 42, 3147−3153). Two of the most potent analogues were the retro-inverso-dipeptide amides l-ArgNO 2 -l-Lys-NH2 (LL) and d-Lys-d-ArgNO 2 −NH2 (DD). To rationalize the common selectivities of LL and DD, it was proposed that in both cases the nitroarginine group binds at the heme binding site, therefore requiring one of these molecules to undergo a 180° flip to accommodate such an interaction. The present studies confirm that the dipeptides indeed bind to holo-nNOS quite similarly from the point of view of the nitroguanidine functionality, supporting the earlier interpretation. The data further suggest that a substantial fraction of the DD epimer is distributed among other binding geometries.

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The binding of the retro-analogue of glutathione disulfide to glutathione reductase.

Cited by 16 publications

References 9 publications

Ajoene Is an Inhibitor and Subversive Substrate of Human Glutathione Reductase and Trypanosoma cruzi Trypanothione Reductase: Crystallographic, Kinetic, and Spectroscopic Studies

Ajoene Is an Inhibitor and Subversive Substrate of Human Glutathione Reductase and Trypanosoma cruzi Trypanothione Reductase: Crystallographic, Kinetic, and Spectroscopic Studies

Glutathione Reductase Turned into Trypanothione Reductase: Structural Analysis of an Engineered Change in Substrate Specificity^,

ENDOR Spectroscopic Evidence for the Geometry of Binding of retro-inverso-N^ω-Nitroarginine-Containing Dipeptide Amides to Neuronal Nitric Oxide Synthase

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The binding of the retro-analogue of glutathione disulfide to glutathione reductase.

Cited by 16 publications

References 9 publications

Ajoene Is an Inhibitor and Subversive Substrate of Human Glutathione Reductase and Trypanosoma cruzi Trypanothione Reductase: Crystallographic, Kinetic, and Spectroscopic Studies

Ajoene Is an Inhibitor and Subversive Substrate of Human Glutathione Reductase and Trypanosoma cruzi Trypanothione Reductase: Crystallographic, Kinetic, and Spectroscopic Studies

Glutathione Reductase Turned into Trypanothione Reductase: Structural Analysis of an Engineered Change in Substrate Specificity,

ENDOR Spectroscopic Evidence for the Geometry of Binding of retro-inverso-Nω-Nitroarginine-Containing Dipeptide Amides to Neuronal Nitric Oxide Synthase

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Glutathione Reductase Turned into Trypanothione Reductase: Structural Analysis of an Engineered Change in Substrate Specificity^,

ENDOR Spectroscopic Evidence for the Geometry of Binding of retro-inverso-N^ω-Nitroarginine-Containing Dipeptide Amides to Neuronal Nitric Oxide Synthase