THE GLUTAMYL BINDING-SITE OF TRYPANOTHIONE REDUCTASE FROM CRITHIDIA-FASCICULATA - ENZYME-KINETIC PROPERTIES OF GAMMA-GLUTAMYL-MODIFIED SUBSTRATE-ANALOGS

Trypanothione reductase, central to the redox defense systems of paras itic trypanosomes and leishmanias, is sufficiently different in its su bstrate-specificity from mammalian glutathione reductase to represent an attractive target for chemotherapeutic intervention. Previous studi es of the physiological substrates trypanothione (N1,N8-bis(glutathion yl)spermidine) and N1-glutathionylspermidine disulphide established th at the spermidine moiety of these substrates can be replaced by the 3- dimethylpropylamide group (N1-glutathionyl-N3-dimethyl-propylamide). W ith this modification, the specificity for the gamma-glutamyl moiety o f the substrate was examined. Kinetic analysis of a series of substrat e analogues indicated that neither the alpha-carboxylate or a-amino fu nctions of the L-gamma-glutamyl group is essential for recognition, si nce this group could be replaced by uncharged benzyloxycarbonyl or t-b utyloxycarbonyl groups with relative catalytic efficiencies (k(cat)/K( m)) of 58 and 11%, respectively, of N1-glutathionyl-N3-dimethylpropyla minedisulphide. Other substitutions are less well tolerated (e.g., bet a-L-aspartyl or aminobutyryl) or not at all (e.g., glutaryl). These fi ndings are discussed in relation to the structural model of TR from Tr ypanosoma congolense. The successful structural replacements achieved have potential application for drug delivery.