Divalent metal requirements for catalysis and stability of the RNA triphosphatase from Trypanosoma cruzi.
RNA triphosphatases act in the first step of the mRNA capping process, removing the gamma-phosphoryl group from the 5' end of nascent RNA. A metal-dependent catalysis is found in the enzymes from trypanosomes and several other lower eukaryotes. This contrasts with the cysteine-dependent activity of the corresponding enzymes of mammals, a difference that points to these enzymes as potential targets for drug design. This work describes the identification, expression, purification, enzyme kinetics, and the role of divalent metal in the ATPase activity of the RNA triphosphatase from Trypanosoma cruzi, the agent of Chagas' disease, and compares it with the previously characterized enzyme from Trypanosoma brucei. Sequence similarity of the T. cruzi enzyme with the RNA triphosphatase of Saccharomyces cerevisiae indicates that a tunnel domain containing the divalent metal forms its active site. Based on enzyme kinetics, circular dichroism, and intrinsic fluorescence analysis, a kinetic mechanism for the ATPase activity of the T. cruzi tunnel triphosphatase is proposed. A single metal is sufficient to interact with the enzyme through the formation of a productive MnATP-enzyme complex, while free ATP inhibits activity. Manganese is also required for the tunnel stability of the T. cruzi enzyme, while the T. brucei homologue remains stable in the absence of metal, as shown for other triphosphatases. These findings may be useful to devise specific triphosphatase inhibitors to the T. cruzi enzyme.
Massayuki Kikuti
Carlos
C
Departamento de Microbiologia, Imunologia e Parasitologia - Universidade Federal de São Paulo, 04023-062 São Paulo, Brazil.
Tersariol
Ivarne Luis S
IL
Schenkman
Sergio
S
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002942:Circular Dichroism; D003067:Coenzymes / Q000378:metabolism; D004795:Enzyme Stability; D005453:Fluorescence; D006358:Hot Temperature; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000201:enzymology; D014349:Trypanosoma cruzi / Q000201:enzymology*
D003067:Coenzymes; D008345:Manganese; D012313:RNA; D000255:Adenosine Triphosphate; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase