TY - JOUR
T1 - Cloning and kinetic characterization of the Trypanosoma cruzi S-adenosylmethionine decarboxylase
AU - Kinch, Lisa N.
AU - Scott, Jerry R.
AU - Ullman, Buddy
AU - Phillips, Margaret A.
N1 - Funding Information:
We thank Dr Jerry E. Manning for providing the T. cruzi cDNA library, Dr Clive Slaughter for the N-terminal sequence analysis of the AdoMetDC subunits, N. Grishin for help with sequence alignments, and H. Brooks for helpful discussion. This work was supported by grants to MAP from the National Institutes of Health (R01 AI34432), the Burroughs Wellcome Fund, the Welch Foundation (I-1257) and the American Heart Association, to LNK from the National Institutes of Health Biophysics Predoctoral Training Program (T32GM08297), and to BU from the National Institutes of Health (R01 AI41622) and the Burroughs Wellcome Fund.
PY - 1999/6/25
Y1 - 1999/6/25
N2 - The gene for S-adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in the biosynthesis of polyamines, has been cloned from a Trypanosoma cruz cDNA library. The cDNA clone contains a 1.1 kb open reading frame predicted to encode a 42 kDa protein that shares 31% sequence identity to the human proenzyme. T. cruzi AdoMetDC expressed and purified from E. coli is auto-catalytically processed into two subunits of 32 kDa (α) and 10 kDa (β). The catalytic activity of the purified recombinant enzyme is activated by the addition of putrescine to the reaction. To determine the effect of putrescine on the kinetics of the reaction, the velocity data collected at various substrate and putrescine concentrations were fit to the rate equation describing a non-essential activator. In the presence of fully saturating putrescine, k(cat) increases by 9-fold over the unactivated rate to 0.06 s-1. The model derived K(m) for AdoMet is 0.05 mM in the absence of putrescine and the model-derived K(d) for putrescine binding to free enzyme is 2.5 mM. The K(m) for AdoMet increases by ~2-fold when the enzyme is fully saturated with putrescine. Unlike human AdoMetDC, cadaverine activates the T. cruzi enzyme to a similar extent as putrescine. Copyright (C) 1999 Elsevier Science B.V.
AB - The gene for S-adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in the biosynthesis of polyamines, has been cloned from a Trypanosoma cruz cDNA library. The cDNA clone contains a 1.1 kb open reading frame predicted to encode a 42 kDa protein that shares 31% sequence identity to the human proenzyme. T. cruzi AdoMetDC expressed and purified from E. coli is auto-catalytically processed into two subunits of 32 kDa (α) and 10 kDa (β). The catalytic activity of the purified recombinant enzyme is activated by the addition of putrescine to the reaction. To determine the effect of putrescine on the kinetics of the reaction, the velocity data collected at various substrate and putrescine concentrations were fit to the rate equation describing a non-essential activator. In the presence of fully saturating putrescine, k(cat) increases by 9-fold over the unactivated rate to 0.06 s-1. The model derived K(m) for AdoMet is 0.05 mM in the absence of putrescine and the model-derived K(d) for putrescine binding to free enzyme is 2.5 mM. The K(m) for AdoMet increases by ~2-fold when the enzyme is fully saturated with putrescine. Unlike human AdoMetDC, cadaverine activates the T. cruzi enzyme to a similar extent as putrescine. Copyright (C) 1999 Elsevier Science B.V.
KW - Polyamines
KW - S-adenosylmethionine decarboxylase
KW - T. cruzi
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U2 - 10.1016/S0166-6851(98)00181-9
DO - 10.1016/S0166-6851(98)00181-9
M3 - Article
C2 - 10413038
AN - SCOPUS:0032998601
SN - 0166-6851
VL - 101
SP - 1
EP - 11
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1-2
ER -