Structure-function analysis of trypanosoma brucei RNA triphosphatase and evidence for a two-metal mechanism Journal Article
| Authors: | Gong, C.; Martins, A.; Shuman, S. |
| Article Title: | Structure-function analysis of trypanosoma brucei RNA triphosphatase and evidence for a two-metal mechanism |
| Abstract: | 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 13 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 (Ki 1.4 μM) that binds more avidly to the active site than the ATP substrate (Km 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. |
| Keywords: | unclassified drug; methylation; nonhuman; protein domain; animal cell; animals; amino acid substitution; phosphatase; rna triphosphatase; enzyme activity; trypanosoma brucei; trypanosoma; acid anhydride hydrolases; structure activity relation; animalia; rna; amino acid sequence; kinetics; enzyme analysis; sequence alignment; amino acid; mutagenesis, site-directed; binding sites; rna stability; alanine; catalysis; amino acids; protein tertiary structure; protozoa; fungi; enzymes; thermostability; metal; metals; rna splicing; chlorella virus; enzyme active site; structure-function analysis; magnesium; polypeptides; polypeptide; competitive inhibition; hydrophilicity; rna analysis; manganese; sedimentation; priority journal; article; structural analysis; two-metal mechanism; tbcet1 triphosphatase; trypanosoma brucei brucei |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 278 |
| Issue: | 51 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2003-12-19 |
| Start Page: | 50843 |
| End Page: | 50852 |
| Language: | English |
| DOI: | 10.1074/jbc.M309188200 |
| PUBMED: | 14525979 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 12 September 2014 -- Source: Scopus |
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