Schizosaccharomyces pombe carboxyl-terminal domain (CTD) phosphatase Fcp1: Distributive mechanism, minimal CTD substrate, and active site mapping Journal Article
| Authors: | Hausmann, S.; Erdjument-Bromage, H.; Shuman, S. |
| Article Title: | Schizosaccharomyces pombe carboxyl-terminal domain (CTD) phosphatase Fcp1: Distributive mechanism, minimal CTD substrate, and active site mapping |
| Abstract: | Schizosaccharomyces pombe Fcp1 is an essential protein serine phosphatase that preferentially dephosphorylates Ser2 of the RNA polymerase II C-terminal domain (CTD) heptad repeat Y1S2P3T 4S5P6S7. Here we show that: (i) Fcp1 acts distributively during the hydrolysis of substrates containing tandem Ser2-PO4 heptads; (ii) the minimal optimal CTD substrate for Fcp1 is a single heptad of phasing S5P6S 7Y1S2P3T4; and (iii) single alanine mutations of flanking residues Tyr1 or Pro 3 result in 6-fold decrements in CTD phosphatase activity. Fcp1 belongs to the DXDX(T/V) family of phosphotransferases that act via an acyl-phosphoenzyme intermediate. An alanine scan of 11 conserved positions of S. pombe Fcp1 identifies Thr174, Tyr237, Thr243, and Tyr249 as important for phosphatase activity. Structure-activity relationships at these positions were determined by introducing conservative substitutions. Our results, together with previous mutational studies, highlight a constellation of 11 amino acids that are conserved in all Fcpl orthologs and likely comprise the active site. |
| Keywords: | unclassified drug; mutation; nonhuman; protein domain; amino acid substitution; carboxy terminal sequence; enzyme activity; structure activity relation; tyrosine; phosphorylation; rna; amino acid sequence; molecular sequence data; sequence homology, amino acid; substrate specificity; amino acid; threonine; binding sites; alanine; amino acids; enzyme substrate complex; rna polymerase ii; hydrolysis; enzymes; enzyme mechanism; schizosaccharomyces; proline; enzyme active site; phosphoprotein phosphatase; schizosaccharomyces pombe; spectrometry, mass, matrix-assisted laser desorption-ionization; dephosphorylation; substitution reactions; protein fcp1; priority journal; article; active site mapping; alanine mutations |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 279 |
| Issue: | 12 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2004-03-19 |
| Start Page: | 10892 |
| End Page: | 10900 |
| Language: | English |
| DOI: | 10.1074/jbc.M312513200 |
| PROVIDER: | scopus |
| PUBMED: | 14701811 |
| DOI/URL: | |
| Notes: | J. Biol. Chem. -- Cited By (since 1996):23 -- Export Date: 16 June 2014 -- CODEN: JBCHA -- Source: Scopus |
Altmetric
Citation Impact
BMJ Impact Analytics
MSK Authors
Related MSK Work