The glutamyl binding site of trypanothione reductase from Crithidia fasciculata: enzyme kinetic properties of γ-glutamyl-modified substrate analogues

Trypanothione reductase, central to the redox defense systems of parasitic trypanosomes and leishmanias, is sufficiently different in its substrate-specificity from mammalian gluthathione reductase to represent an attractive target for chemotherapeutic intervention. Previous studies of the physiological substrates trypanothione (N¹, N⁸-bis(glutathionyl)spermidine) and N¹-glutathionylspermidine disulphide established that spermidine spermidine moiety of these substrates can be replaced by the 3-dimethyl-propylamide group (N¹-glutathionyl-N³-dimethyl-propylamide). With this modification, the specificity for the γ-glutamyl moiety of the substrate was examined. Kinetic analysis of a series of substrate analogues indicated that neither the α-carboxylate or α-amino functions of the l-γ-glutamyl group is essential for recognition, since this group could be replaced by uncharged benzyloxycarbonyl or t-butyloxycarbonyl groups with relative catalytic efficiencies (k_cat/K_m) of 58 and 11%, respectively, of N¹-glutathionyl-N³-dimethylpropylaminedisulphide. Other substitutions are less well tolerated (e.g., β-l-aspartyl or aminobutyryl) or not at all (e.g., glutaryl). These findings are discussed in relation to the structural model of TR from Trypanosoma congolense. The successful structural replacements achieved have potential application for drug delivery.