CHARACTERIZATION OF THE PEPTIDE SUBSTRATE-SPECIFICITY OF GLUTATHIONYLSPERMIDINE SYNTHETASE FROM CRITHIDIA-FASCICULATA

Trypanothione, a metabolite specific to trypanosomatid parasites, is e nzymatically synthesized from spermidine and glutathione by the consec utive action of the ATP-dependent carbon-nitrogen ligases, glutathiony lspermidine synthetase and trypanothione synthetase. As part of our pr ogramme aimed at developing inhibitors of these enzymes, we have synth esized a series of analogues of glutathione (gamma-L-Glu-L-Cys-Gly) an d tested them as substrates or inhibitors of glutathionylspermidine sy nthetase. Recognition at the gamma-glutamyl moiety appears to be essen tial, as any modification of this part of glutathione results in a tot al loss of activity as a substrate. Alkylation of the thiol side chain of cysteine with methyl, ethyl or propyl groups yields analogues with catalytic efficiencies (k(cat)/K-m) as substrates equivalent to or be tter than glutathione. In contrast, the bulkier S-butyl analogue Subst itution of L-Cys by amino acids with an alkyl side-chain is also well tolerated giving relative catalytic efficiencies of 1.1 and 1.5 for pe ptide analogues containing L-Val and L-Ile respectively. Other analogu es, where the bulk of the alkyl chain is increased further (as in L-Le u or L-Phe) or where the glycine moiety is replaced with L-Ala, are in hibitors rather than substrates. Copyright (C) 1997 Elsevier Science B .V.