Cloning and kinetic characterization of the Trypanosoma cruzi S-adenosylmethionine decarboxylase.
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 (alpha) and 10 kDa (beta). 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 Km for AdoMet is 0.05 mM in the absence of putrescine and the model-derived Kd for putrescine binding to free enzyme is 2.5 mM. The Km for AdoMet increases by alpha 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.
Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 75235, USA.
L N
LN
Kinch
J R
JR
Scott
B
B
Ullman
M A
MA
Phillips
D012437:Adenosylmethionine Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002103:Cadaverine / Q000378:metabolism; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011700:Putrescine / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D016054:DNA, Protozoan; D011994:Recombinant Proteins; D012437:Adenosylmethionine Decarboxylase; D002103:Cadaverine; D011700:Putrescine