Regulation of Trypanosoma Cruzi S-Adenosylmethionine Decarboxylase Activity

Chagas' disease, caused by Trypanosoma cruzi, is a major health problem for which there is no good chemotherapy. Development of new drugs is crucial. One strategy is to develop inhibitors of enzymes in essential metabolic pathways. Trypanosomes need polyamines to survive; thus, the enzymes in the polyamine biosynthetic pathway are targets for drug development. Characterization of S-adenosylmethionine decarboxylase (the rate-limiting enzyme in the pathway) activity in trypanosomes and comparison to human AdoMetDC will aid the discovery of novel, species specific inhibitors. I have characterized putrescine stimulation of T. cruzi AdoMetDC activity as well as the allostery between the putrescine binding site and the active site of the enzyme. Putrescine is capable of stimulating T. cruzi AdoMetDC decarboxylation but not proenzyme processing. This contrasts with the human enzyme, for which processing and decarboxylation are both modestly stimulated by putrescine. R13, a Leu in the human enzyme, was shown to be essential for processing in the T. cruzi enzyme. Additionally, D174 is an important binding determinant for putrescine in both species of AdoMetDC. Other mutations near the putative putrescine site (S111R and F285H) indicate that the putrescine binding site for T. cruzi AdoMet DC is more surface-exposed than the human enzyme. Putrescine stimulates decarboxylase activity 41 fold for T. cruzi AdoMetDC (compared to 1.7 fold for the human enzyme). This stimulation is achieved through allosteric binding to a site approximately 15 ?ay from the active site. To examine this allostery, we used putrescine and active site mutations as well as an active site inhibitor, CGP 40215. This inhibitor unexpectedly exhibited noncompetitive inhibition with a Ki of 32muM and 3muM in the absence and presence of putrescine, respectively (Ki for the human enzyme is 10nM and 6nM). Residues important for communication between the two sites include L221, H5, D174, S111 and F285. L221, a Thr residue in the human enzyme, is partially responsible for some of the species specificity as the Ki for L221T was 800nM. The differences in putrescine stimulation and CGP 40215 inhibition indicate development of species specific inhibitors is possible and could be an effective strategy for novel antitrypanosomal chemotherapy.