Biosynthesis of the trypanosomatid metabolite trypanothione: purification and characterization of trypanothione synthetase from Crithidia fasciculata.
Trypanothione synthetase from Crithidia fasciculata has been purified ca. 14,500-fold to homogeneity in an overall yield of 40%. The pure enzyme catalyzed the synthesis of N1- and N8-glutathionylspermidine and N1,N8-bis(glutathionyl)spermidine (trypanothione) from ATP/magnesium, glutathione (GSH), and spermidine, N1- and N8-glutathionylspermidines being intermediates of trypanothione synthesis. The enzyme showed a sharp pH optimum of 7.5-7.75 for the synthesis of both mono- and diglutathionylspermidine conjugates. It was highly specific for its physiological substrates ATP/Mg2+, GSH, spermidine, and N1- and N8-glutathionylspermidine with Km values of 400 microM, 914 microM, 1.07 mM, 20 microM, and 7 microM, respectively. Trypanothione synthetase was active in the monomeric form with Mr = 87,000 and absorption maxima lambda max = 225 and 280 nm (A280/A260 = 1.85). Trypanothione synthetase is a new member of the ATP-dependent class of ligases which form amide linkage with concomitant production of ADP and orthophosphate.
Henderson
G B
GB
Laboratory of Medical Biochemistry, Rockefeller University, New York, New York 10021.
Yamaguchi
M
M
Novoa
L
L
Fairlamb
A H
AH
Cerami
A
A
D019732:Amide Synthases; D000818:Animals; D002845:Chromatography; D003421:Crithidia / Q000201:enzymology*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000097:blood; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification*; D008970:Molecular Weight; D013095:Spermidine / Q000031:analogs & derivatives* / Q000097:blood
C044809:trypanothione; D008025:Ligases; D019732:Amide Synthases; C064407:trypanothione synthetase; D005978:Glutathione; D013095:Spermidine