TY - JOUR
T1 - Biosynthesis of the Trypanosomatid Metabolite Trypanothione
T2 - Purification and Characterization of Trypanothione Synthetase from Crithidia fasciculated
AU - Henderson, Graeme B.
AU - Yamaguchi, Miyuki
AU - Novoa, Louis
AU - Fairlamb, Alan H.
AU - Cerami, Anthony
PY - 1990/4/24
Y1 - 1990/4/24
N2 - 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-gluta-thionylspermidine 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 μM, 914 μM, 1.07 mM, 20 μM, and 7 μM, respectively. Trypanothione synthetase was active in the monomeric form with Mr = 87 000 and absorption maxima λmax = 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.
AB - 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-gluta-thionylspermidine 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 μM, 914 μM, 1.07 mM, 20 μM, and 7 μM, respectively. Trypanothione synthetase was active in the monomeric form with Mr = 87 000 and absorption maxima λmax = 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.
UR - http://www.scopus.com/inward/record.url?scp=0025273891&partnerID=8YFLogxK
U2 - 10.1021/bi00468a019
DO - 10.1021/bi00468a019
M3 - Article
C2 - 2354163
AN - SCOPUS:0025273891
SN - 0006-2960
VL - 29
SP - 3924
EP - 3929
JO - Biochemistry
JF - Biochemistry
IS - 16
ER -