Defining the active site of Schizosaccharomyces pombe C-terminal domain phosphatase Fcp1 Journal Article
| Authors: | Hausmann, S.; Shuman, S. |
| Article Title: | Defining the active site of Schizosaccharomyces pombe C-terminal domain phosphatase Fcp1 |
| Abstract: | Fcp1 is an essential protein serine phosphatase that dephosphorylates the C-terminal domain (CTD) of RNA polymerase II. By testing the effects of serial N- and C-terminal deletions of the 723-amino acid Schizosaccharomyces pombe Fcp1, we defined a minimal phosphatase domain spanning amino acids 156-580. We employed site-directed mutagenesis (introducing 24 mutations at 14 conserved positions) to locate candidate catalytic residues. We found that alanine substitutions for Arg223, Asp258, Lys280, Asp297, and Asp298 abrogated the phosphatase activity with either p-nitrophenyl phosphate or CTD-PO4 as substrates. Structure-activity relationships were determined by introducing conservative substitutions at each essential position. Our results, together with previous mutational studies, highlight a constellation of seven amino acids (Asp170, Asp172, Arg223, Asp258, Lys280, Asp297, and Asp298) that are conserved in all Fcp1 orthologs and likely comprise the active site. Five of these residues (Asp170, Asp172, Lys280, Asp297, and Asp298) are conserved at the active site of T4 polynucleotide 3′-phosphatase, suggesting that Fcp1 and T4 phosphatase are structurally and mechanistically related members of the DXD phosphotransferase superfamily. |
| Keywords: | controlled study; unclassified drug; gene deletion; mutation; dose response; nonhuman; molecular genetics; protein domain; metabolism; amino acid substitution; phosphatase; carboxy terminal sequence; enzymology; dose-response relationship, drug; structure activity relation; chemistry; amino acid sequence; molecular sequence data; sequence homology, amino acid; nucleotide sequence; nucleic acids; binding site; amino acid; mutagenesis, site-directed; protein structure, tertiary; binding energy; binding sites; dna mutational analysis; alanine; catalysis; amino acids; sequence homology; enzyme structure; site directed mutagenesis; catalytic domain; protein tertiary structure; aspartic acid; enzymes; lysine; arginine; schizosaccharomyces; enzyme active site; phosphoprotein phosphatase; schizosaccharomyces pombe; organophosphorus compounds; nitrophenols; fungal enzyme; organophosphorus compound; active sites; nitrophenol; protein fcp1; priority journal; article; polynucleotide 3' phosphatase; carboxy terminal domain phosphatase; carboxy-terminal domain phosphatase; nitrophenyl phosphate |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 278 |
| Issue: | 16 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2003-04-18 |
| Start Page: | 13627 |
| End Page: | 13632 |
| Language: | English |
| DOI: | 10.1074/jbc.M213191200 |
| PUBMED: | 12556522 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 12 September 2014 -- Source: Scopus |
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