Trypanothione reductase from Trypanosoma cruzi. Purification and characterization of the crystalline enzyme.
The structural differences between trypanothione reductase of Trypanosoma cruzi and human glutathione reductase, an enzyme of known three-dimensional structure, offer an opportunity for rational drug design against Chagas' disease. As a first step in the analysis of the parasite enzyme we report its purification and characterization. 2.2 mg trypanothione reductase was extracted from 33 g wet weight of cultured epimastigotes or from 4 g lyophilized cells. The flavoenzyme was purified 2400-fold to homogeneity in three steps with an overall yield of 45%. The enzyme is a dimer with a subunit Mr of 50,000. Using NADPH (Km = 5 microM) and trypanothione disulfide (Km = 45 microM) as substrates, a turnover number of 14,200 min-1 was estimated. Trypanothione reductase, the parasite enzyme, and glutathione reductase, the host enzyme, exhibit mutually exclusive specificities for their respective disulfide substrates. When screening cell cultures or column eluates for the presence of trypanothione reductase, a microassay based on Ellman's reagent as indicator was used. A mixture of regioisomeric glutathionylspermidine disulfides isolated from Escherichia coli served as substrate in this microassay. Experimentally, the catalytic cycle of the enzyme can be subdivided into the half-reactions Eox + NADPH + H+----EH2 + NADP+, and EH2 + trypanothione disulfide----Eox + dihydrotrypanothione. This is also true for the crystallized enzyme in the presence of 2 M (NH4)2SO4. The spectral properties of trypanothione reductase both in the oxidized form (Eox) and in the two-electron-reduced form (EH2) closely resemble those of human glutathione reductase. Both proteins contain a flavin and a redox-active disulfide at the catalytic site. After reduction of Eox to EH2, trypanothione reductase can be inactivated by specifically alkylating one of the nascent active-site thiols.
D000818:Animals; D003460:Crystallization; D005980:Glutathione Reductase / Q000032:analysis; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D009249:NADP / Q000378:metabolism; D009547:Nifurtimox / Q000494:pharmacology; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D005980:Glutathione Reductase; D009547:Nifurtimox