Deletion of the parasite-specific insertions and mutation of the catalytic triad in glutathione reductase from chloroquine-sensitive Plasmodium falciparum 3D7

The flavoenzyme glutathione reductase (GR; NADPH + glutathione disulphide + H+ --> NADP(+) + 2 glutathione-SH) of Plasmodium falciparum is a promising drug target against tropical malaria. As P. falciprum genes are assumed to be highly polymorphic we have cloned and expressed the GR cDNA of the chloroquine-sensitive strain 3D7. In comparison to the known GR of the chloroquine-resistant K1 strain there are three base exchanges all of them leading to amino acid substitutions (residues 281, 285 and 335), The catalytic efficiency k(cat)/K-m of the 3D7 enzyme is 5-fold lower than for the K1 enzyme. In contrast. vis-a-vis the drugs carmustine, methylene blue and fluorophenyliso-alloxazine the two enzyme species exhibited identical inhibition kinetics. Two- structural motifs which are specific for P, falciparum GR were studied by mutational deletion analysis of 3D7 GR. Loop 126-138 appears to be important for folding and stability of the enzyme, whereas the subdomain 318-350 was found to be involved in FAD-binding. The subdomain has no major influence on the known functions of the catalytic triad Cys-40, Cys-45 and Mis-485'. Flavin absorption spectroscopy of inactive point mutants showed that Cys-45 forms a thiolate charge transfer complex and Cys-40 is the interchange thiol, which reduces glutathione disulphide. The mutant His-485 --> Gin had a normal k'(m) for glutathione disulphide reduction but only 0.8% residual catalytic activity when compared with wild-type GR, which confirms its function as an acid/base catalyst. The parasite-specific domains in combination with the reactive catalytic residues appear to be a suitable target matrix for inhibiting GR in vivo. (C) 2000 Elsevier Science B.V, All rights reserved.