Structure-function analysis of Trypanosoma brucei RNA triphosphatase and evidence for a two-metal mechanism

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 beta 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 ( (76)WKGRRARKT(84)) 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 degrees 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 muM) that binds more avidly to the active site than the ATP substrate ( K-m 25 muM). 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.