Substrate specificity of the flavoprotein trypanothione disulfide reductase from Crithidia fasciculata

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Abstract
The substrate specificity of the trypanosomatid enzyme trypanothione reductase has been studied by measuring the ability of the enzyme to reduce a series of chemically synthesized cyclic and acyclic derivatives of N1, N8-bis(glutathionyl)spermidine disulfide (trypanothione). Kinetic analysis of the enzymatic reduction of these synthetic substrates indicates that the mutually exclusive substrate specificity observed by the NADPH-dependent trypanothione disulfide reductase and the related flavoprotein glutathione disulfide reductase is due to the presence of a spermidine binding site in the substrate binding domain of trypanothione reductase. Trypanothione reductase will reduce the disulfide form of N1-monoglutathionylspermidine and also the mixed disulfide of N1-monoglutathionylspermidine and glutathione. The Michaelis constants for these reactions are 149 .mu.M and 379 .mu.M, respectively. Since the disulfide form of N1-monoglutathionyspermidine and the mixed disulfide of N1-monoglutathionylspermidine and glutathione could be formed in trypanosomatids, the binding constants and turnover numbers for the enzymatic reduction of these acyclic disulfides are consistent with these being potential alternative substrates for trypanothione reductase in vivo.