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 Ser(2) of the RNA polymerase II C-terminal domain (CTD) heptad repeat Y(1)S(2)P(3)T(4)S(5)P(6)S(7). Here we show that: (i) Fcp1 acts distributively during the hydrolysis of substrates containing tandem Ser(2)-PO(4) heptads; (ii) the minimal optimal CTD substrate for Fcp1 is a single heptad of phasing S(5)P(6)S(7)Y(1)S(2)P(3)T(4); and (iii) single alanine mutations of flanking residues Tyr(1) 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 Thr(174), Tyr(237), Thr(243), and Tyr(249) 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 Fcp1 orthologs and likely comprise the active site.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Hausmann
authors.forename: Hediye
authors.initials: H
authors.lastname: Erdjument-Bromage
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D012568:Schizosaccharomyces / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D010749:Phosphoprotein Phosphatases; C116022:carboxy-terminal domain phosphatase