Glutathione reductase (NADPH+GSSG+H+-->NADP(+) + 2GSH) is a homodimeric flavoenzyme of known geometry. Each subunit contains four well-defined domains and contributes essential residues to the active sites; consequently, the monomer is expected to be inactive. As part of our program to develop dimerization inhibitors of human glutathione reductase (hGR) as antimalarial agents, we mutagenized the residues 446 and 447 which, together with their counterparts on the other subunit, represent the tightest contact between the subunits [Karplus, P. A., & Schulz, G. E. (1987) J. Mol. Biol. 195, 701-729]. Wild-type human glutathione reductase and mutants of this protein were produced in plasmid-transformed Escherichia coli SG5 cells. Active enzyme species, namely, wild-type hGR, N-terminally truncated delta(1-15)hGR, and the point mutant F447P-hGR, were purified by 2',5'-ADP-Sepharose chromatography and crystallization. Inactive mutants such as G446E-hGR or the double mutants G446E/F447P-hGR and G446P/F447P-hGR were isolated by immunoadsorption chromatography. G446E/F447P-hGR was studied in detail. This mutant behaved like a poorly folded monomeric protein, as indicated by the following properties: absence of the intersubunit disulfide bridge, Cys90-Cys90'; failure to bind FAD; failure to bind NADPH and analogues thereof; a short half-life (< 4 min) in E. coli cells; and high susceptibility to trypsin in vitro. The results suggest that the sequence around G446 can control dimerization as well as domain folding. This is unexpected since the FAD-binding domain and the NADPH-binding domain occur in many different enzymes and have been regarded as autonomous folding units.(ABSTRACT TRUNCATED AT 250 WORDS)
Trypanothione reductase of Trypanosoma cruzi is a key enzyme in the antioxidant metabolism of the parasite. Here we report on the enzymic and pharmacological properties of trypanothione reductase using glutathionylspermidine disulfide as a substrate.1. Both pH optimum (7.5) and the ionic strength optimum (at 30 mM) are unusually narrow for this enzyme. 40 mM Hepes, 1 mM EDTA, pH 7.5 was chosen as a standard assay buffer because in this system the k,,,/K, ratio had the highest values for both natural substrates, glutathionylspermidine disulfide (2.65 x lo6 M-' s-' 1 and trypanothione disulfide (4.63 x lo6 M-' s f l 1.2. Using the standardized assay, trypanothione reductase and the phylogenetically related host enzyme, human glutathione reductase, were studied as targets of inhibitors. Both enzymes, in their NADPH-reduced forms, were irreversibly modified by the cytostatic agent, 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU). Nifurtimox, the drug used in the treatment of Chagas' disease, is a stronger inhibitor of glutathione reductase (Ki = 40 pM) than of trypanothione reductase (ICso = 200 pM).3. Of the newly synthesized trypanocidal compounds [Henderson, G. B., Ulrich, P., Fairlamb, A. H., Rosenberg, I., Pereira, M., Sela, M. & Cerami, A. (1988) Proc. Natl Acad. Sci., 85, 5374-53781 a nitrofuran derivative, 2-(5 -nitro -2-furanylmethy1idene)-N,W-[1,4-piperazinediylbis (1,3 -propanediyl)]bishydrazinecarboximidamide tetrahydrobromide, was found to be a better inhibitor for trypanothione reductase (Ki = 0.5 pM) than for glutathione reductase (ICso = 10 pM). A naphthoquinone derivative, 2,3-bis[3-(2-amidinohydrazono)-butyl]-l,4-naphthoquinone dihydrochloride, turned out to be both an inhibitor = 1 pM) and an NADPHoxidation-inducing substrate (K, = 14 pM). This effect was not observed with human glutathione reductase. Such compounds which lead to oxidative stress by more than one mechanism in the parasite are promising starting points for drug design based on the three-dimensional structures of glutathione and trypanothione reductases. Enzymes. Trypanothione reductase (EC 1.6.4.-); glutathione reductase (EC 1.6.4.2). donovani) and oriental sore (Leishmania tropica) are caused by these protozoa.In contrast to all other known eukaryotes [l, 21 Trypanosomatidae do not possess the flavoenzyme glutathione reductase that is responsible for a high intracellular glutathione/ glutathione disulfide ratio. The thiol metabolism of trypanosomes and leishmanias is based on conjugates between glutathione and spermidine, namely trypanothione [3] and glutathionylspermidine [4]. These thiols are kept in the reduced state by trypanothione reductase which catalyzes the following reactions: trypanothione disulfideTrypanothione reductase is an FAD-cystine oxidoreductase which has been purified from the insect parasite Crithidia fasciculata [5] and, in crystalline form, from T . cruzi [6]. The DNA sequence is known for the enzyme from T. congolense [7].In vitro studies on trypanothione reductase are hampered by the fact that the natural dis...
Tyr114 and Tyr197 are highly conserved residues in the active site of human glutathione reductase, Tyr114 in the glutathione disulfide (GSSG) binding site and Tyr197 in the NADPH site. Mutation of either residue has profound effects on catalysis. Y197S and Y114L have 17% and 14% the activity of the wild-type enzyme, respectively. Mutation of Tyr197, in the NADPH site, leads to a decrease in Km for GSSG, and mutation of Tyr114, in the GSSG site, leads to a decrease in Km for NADPH. This behavior is predicted for enzymes operating by a ping-pong mechanism where both half-reactions partially limit turnover. Titration of the wild-type enzyme or Y114L with NADPH proceeds in two phases, Eox to EH2 and EH2 to EH2-NADPH. In contrast, Y197S reacts monophasically, showing that excess NADPH fails to enhance the absorbance of the thiolate-FAD charge-transfer complex, the predominant EH2 form of glutathione reductase. The reductive half-reactions of the wild-type enzyme and of Y114L are similar; FAD reduction is fast (approximately 500 s-1 at 4 degreesC) and thiolate-FAD charge-transfer complex formation has a rate of 100 s-1. In Y197S, these rates are only 78 and 5 s-1, respectively. The oxidative half-reaction, the rate of reoxidation of EH2 by GSSG, of the wild-type enzyme is approximately 4-fold faster than that of Y114L. These results are consistent with Tyr197 serving as a gate in the binding of NADPH, and they indicate that Tyr114 assists the acid catalyst His467'.
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