Substrate Specificity of Trypanothione Reductase

Abstract

Trypanothione redutase, one of the family of flavin-dependent disulfide oxidoreductases, catalyses the reduction of trypanothione disulfide [N1,N1'-bis(glutathionyI)spermidine] and related glutathionyl-polyamine disulfides. A series of subtly different, designed substrate analogues based on trypanothione were prepared by means of a solid-phase approach and used to study the catalytic efficiency of the parasitic enzyme. Kinetic analysis showed that the size of the polyamine bridge was relatively unimportant, for catalysis, while replacement of the ammonium bridge was much more dramatic. The highly charged glutathionylspermidine disulfide had the largest K,, of all the substrates tested. N'-acetylcysteinylglycinyl-I\P-ghtathionyl spermidine and N'-glutathionyl-NH-acetylcysteinylglycinylspermidine both showed a tenfold reduction in catalytic efficiency compared with trypanothione.Keywords: trypanothione reductase ; substrate specificity ; analogue.Trypanothione reductase is an enzyme unique to the parasitic protozoa known as the trypanosomes and leishmania [ l , 21. Its role in the maintenance of a reducing cellular environment within these parasites, and its specificity for its substrate N',Wbis(glutathiony1)spermidine (trypanothione) compared with that of the host metabolite glutathione [3, 41, have led to trypanothione reductase being an attractive target for inhibition and potential antiparasitic-drug development [5 -121. This potential has led to the cloning of the genes for trypanothione reductase from at least four sources [ 13 -171 and the protein has been expressed at high levels in fkherichia coli [6,15, 181, thus allowing the full arsenal of physical and biochemical techniques to be unleashed in the study of this enzyme. High-resolution X-ray crystal structures 119-221, molecular modelling 161, extensive mutagenesis studies [3, 231, inhibitor design and synthesis 15-10] and detailed kinetic analysis [24] have been used in the study of this enzyme and have, in addition to studies on the analogous human enzyme glutathione reductase [25-291, allowed a detailed understanding of the mechanism and substrate specificities of trypanothione reductase and glutathione reductase. However, since an early study by Henderson et al. 1291, few studies have reported on the specific substrate requirements of trypanothione reductase [30-321. The aim of the present work was to probe the molecular details of substrate recognition in trypanothione reductase, answering questions concerning the importance of the unsymmetrical spermidine bridge and the binding of the two glutamic acid side chains, which, as determined by X-ray crystal-structure analysis, bind in strikingly different conformations (191. MATERIALS AND METHODSTrypanosoma cruzi trypanothione reductase wac a kind gift from Professor Alan Fairlamb. Protein concentrations were deCorresporzdence to M. Bradley, Department of Chemistry, University of Southampton, Southampton. SO17 1 BJ, UK Ahhreviafions. Boc, f-butoxycarbonyl, BLI', t-butyl ; ES, electrospray ; GR, gluta...

Trypanothione as a Target in the Design of Antitrypanosomal and Antileishmanial Agents

“…Trypanothione homologues like homotrypanothione and nortrypanothione are however rather efficient substrates with k cat /K m only slightly lower than trypanothione [80]. Deleting one glutamic acid residue reduce the efficiency: N 1 -acetylcysteinylglycyl-N 8 -glutathionylspermidine and N 8 -acetylcysteinylglycyl-N 1 -glutathionylspermidine are 10 times less efficicient as a substrate than trypanothione.…”

Discovery of 2-iminobenzimidazoles as a new class of trypanothione reductase inhibitor by high-throughput screening

“…2). 11 GR has a hydrophilic, positively charged region in its active site that interacts with the glycine carboxylates of glutathione disulfide, while TR has a larger binding site with a negatively charged region with which the spermidine moiety of trypanothione disulfide binds. 12 The absence of TR from the mammalian host and the sensitivity of trypanosomatids to oxidative stress makes TR an attractive target for trypanosomiasis therapeutics.…”

Trypanothione Reductase High-Throughput Screening Campaign Identifies Novel Classes of Inhibitors with Antiparasitic Activity

“…TR and human GR have similar catalytic mechanisms; 14 of the 19 amino acid residues close to the binding site are conserved. However, they are specific to their respective disulfide substrates (36) (Fig. 1B).…”

“…1C). Studies of the substrate specificity of TR have found that the active site tolerates noncognate substrate architecture and removal of the disulfide moiety (36), such that a number of nonreducible acyclic and cyclic substrate analogues (13) display TR inhibition. Polyamine-and peptide-based TR inhibitors are generally structurally related to spermine and spermidine and have been shown to be effective competitive inhibitors (32).…”