Divalent metal requirements for catalysis and stability of the RNA triphosphatase from Trypanosoma cruzi

Divalent metal requirements for catalysis and stability of the RNA triphosphatase from Trypanosoma cruzi
Received 28 January 2006; revised 27 June 2006; accepted 28 June 2006. Available online 20 July 2006.
Carlos Massayuki Kikutia, Ivarne Luis S. Tersariolb and Sergio Schenkmana
Molecular and Biochemical Parasitology
Volume 150, Issue 1 , November 2006
Copyright ? 2006 Elsevier B.V. All rights reserved.
aDepartamento de Microbiologia, Imunologia e Parasitologia - Universidade Federal de S?o Paulo, R. Botucatu 862 8a, 04023-062 S?o Paulo, Brazil
bCentro Interdisciplinar de Investiga??o Bioqu?mica, Universidade de Mogi das Cruzes, 08701-970 Mogi das Cruzes, Brazil
RNA triphosphatases act in the first step of the mRNA capping process, removing the ?-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.
Keywords: Trypanosoma cruzi; Trypanosoma brucei; RNA triphosphatase; ATPase; Kinetics; Circular dichroism; Fluorescence; Structure; Manganese
Abbreviations: CD, circular dichroism; Cet1, RNA triphosphatase from Saccharomyces cerevisiae; Ea, energy of activation; TbCet1, RNA triphosphatase from Trypanosoma brucei; TcCet1, RNA triphosphatase from Trypanosoma cruzi
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