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J. Biol. Chem., Vol. 278, Issue 51, 50843-50852, December 19, 2003

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Structure-Function Analysis of Trypanosoma brucei RNA Triphosphatase and Evidence for a Two-metal Mechanism^*

Chunling Gong, Alexandra Martins, and Stewart Shuman

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

Trypanosoma brucei RNA triphosphatase TbCet1 is a 252-amino acid polypeptide that catalyzes the first step in mRNA cap formation. By performing an alanine scan of TbCet1, we identified six amino acids that are essential for triphosphatase activity (Glu-52, Arg-127, Glu-168, Arg-186, Glu-216, and Glu-218). These results consolidate the proposal that protozoan, fungal, and Chlorella virus RNA triphosphatases belong to a single family of metal-dependent NTP phosphohydrolases with a unique tunnel active site composed of eight strands. Limited proteolysis of TbCet1 suggests that the hydrophilic N terminus is surface-exposed, whereas the catalytic core domain is tightly folded with the exception of a protease-sensitive loop (⁷⁶WKGRRARKT⁸⁴) between two of the putative tunnel strands. The catalytic domain of TbCet1 is extraordinarily thermostable. It remains active after heating for 2 h at 75 °C. Analysis by zonal velocity sedimentation indicates that TbCet1 is a monomeric enzyme, unlike fungal RNA triphosphatases, which are homodimers. We show that tripolyphosphate is a potent competitive inhibitor of TbCet1 (K_i 1.4 µM) that binds more avidly to the active site than the ATP substrate (K_m 25 µM). We present evidence of synergistic activation of the TbCet1 triphosphatase by manganese and magnesium, consistent with a two-metal mechanism of catalysis. Our findings provide new insight to the similarities (in active site tertiary structure and catalytic mechanism) and differences (in quaternary structure and thermal stability) among the different branches of the tunnel enzyme family.

Received for publication, August 19, 2003 , and in revised form, September 27, 2003.

^* This work was supported by National Institutes of Health Grant GM52470 and a New Initiatives Research Award from the Burroughs Wellcome Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. E-mail: s-shuman{at}ski.mskc.org.

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