The glutamyl binding site of trypanothione reductase from Crithidia fasciculata: enzyme kinetic properties of gamma-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 glutathione 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 the 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 gamma-glutamyl moiety of the substrate was examined. Kinetic analysis of a series of substrate analogues indicated that neither the alpha-carboxylate or alpha-amino functions of the L-gamma-glutamyl group is essential for recognition, since this group could be replaced by uncharged benzyloxycarbonyl or t-butyloxycarbonyl groups with relative catalytic efficiencies (kcat/Km) of 58 and 11%, respectively, of N1-glutathionyl-N3-dimethylpropylaminedisulphide. Other substitutions are less well tolerated (e.g., beta-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.
el-Waer
A F
AF
Department of Pharmacy, University of Manchester, UK.
Smith
K
K
McKie
J H
JH
Benson
T
T
Fairlamb
A H
AH
Douglas
K T
KT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016831:Crithidia fasciculata / Q000201:enzymology*; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D005978:Glutathione / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D013095:Spermidine / Q000031:analogs & derivatives / Q000737:chemistry; D013379:Substrate Specificity
D005971:Glutamates; D018698:Glutamic Acid; C044809:trypanothione; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D005980:Glutathione Reductase; D005978:Glutathione; D013095:Spermidine