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 Trypanoso ma cruz cDNA library. The cDNA clone contains a 1.1 kb open reading frame p redicted to encode a 42 kDa protein that shares 31% sequence identity to th e human proenzyme. T. cruzi AdoMetDC expressed and purified from E. coli is auto-catalytically processed into two subunits of 32 kDa (cc) and 10 kDa ( beta). The catalytic activity of the purified recombinant enzyme is activat ed by the addition of putrescine to the reaction. To determine the effect o f putrescine on the kinetics of the reaction, the velocity data collected a t various substrate and putrescine concentrations were fit to the rate equa tion describing a non-essential activator. In the presence of fury saturati ng 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 putre scine and the model-derived K-d for putrescine binding to free enzyme is 2. 5 mM. The K-m for AdoMet increases by approximate to 2-fold when the enzyme is fully saturated with putrescine. Unlike human AdoMetDC, cadaverine acti vates the T. cruzi enzyme to a similar extent as putrescine. (C) 1999 Elsev ier Science B.V. All rights reserved.