TY - JOUR
T1 - Characterization of the peptide substrate specificity of glutathionylspermidine synthetase from Crithidia fasciculata
AU - De Craecker, Sofie
AU - Verbruggen, Christophe
AU - Rajan, Padinchare K.
AU - Smith, Keith
AU - Haemers, Achiel
AU - Fairlamb, Alan H.
N1 - Funding Information:
This work was supported by grants from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), the Belgian Nationaal Fonds voor Wetenschappelijk Onderzoek, the Geconcerteerde Onderzoeksactie 92/94-09 of the Flemish Community, the Wellcome Trust and the British Council.
PY - 1997/1
Y1 - 1997/1
N2 - Trypanothione, a metabolite specific to trypanosomatid parasites, is enzymatically synthesized from spermidine and glutathione by the consecutive action of the ATP-dependent carbon-nitrogen ligases, glutathionylspermidine synthetase and trypanothione synthetase. As part of our programme aimed at developing inhibitors of these enzymes, we have synthesized a series of analogues of glutathione (γ-L-Glu-L-Cys-Gly) and tested them as substrates or inhibitors of glutathionylspermidine synthetase. Recognition at the γ-glutamyl moiety appears to be essential, as any modification of this part of glutathione results in a total loss of activity as a substrate. Alkylation of the thiol side chain of cysteine wilh methyl, ethyl or propyl groups yields analogues with catalytic efficiencies (kcat/Km) as substrates equivalent to or better than glutathione. In contrast, the bulkier S-butyl analogue was a much poorer substrate. Substitution 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 peptide analogues containing L-Val and L-Ile respectively. Other analogues, where the bulk of the alkyl chain is increased further (as in L-Leu or L-Phe) or where the glycine moiety is replaced with L-Ala, are inhibitors rather than substrates.
AB - Trypanothione, a metabolite specific to trypanosomatid parasites, is enzymatically synthesized from spermidine and glutathione by the consecutive action of the ATP-dependent carbon-nitrogen ligases, glutathionylspermidine synthetase and trypanothione synthetase. As part of our programme aimed at developing inhibitors of these enzymes, we have synthesized a series of analogues of glutathione (γ-L-Glu-L-Cys-Gly) and tested them as substrates or inhibitors of glutathionylspermidine synthetase. Recognition at the γ-glutamyl moiety appears to be essential, as any modification of this part of glutathione results in a total loss of activity as a substrate. Alkylation of the thiol side chain of cysteine wilh methyl, ethyl or propyl groups yields analogues with catalytic efficiencies (kcat/Km) as substrates equivalent to or better than glutathione. In contrast, the bulkier S-butyl analogue was a much poorer substrate. Substitution 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 peptide analogues containing L-Val and L-Ile respectively. Other analogues, where the bulk of the alkyl chain is increased further (as in L-Leu or L-Phe) or where the glycine moiety is replaced with L-Ala, are inhibitors rather than substrates.
KW - Crithidia fasciculata
KW - glutathione-binding site
KW - glutathionylspermidine synthetase
KW - kinetic analysis
KW - substrate specificity
KW - trypanothione
UR - http://www.scopus.com/inward/record.url?scp=0031054199&partnerID=8YFLogxK
U2 - 10.1016/S0166-6851(96)02778-8
DO - 10.1016/S0166-6851(96)02778-8
M3 - Article
C2 - 9041518
AN - SCOPUS:0031054199
SN - 0166-6851
VL - 84
SP - 25
EP - 32
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1
ER -