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J. Biol. Chem., Vol. 279, Issue 12, 10892-10900, March 19, 2004

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Schizosaccharomyces pombe Carboxyl-terminal Domain (CTD) Phosphatase Fcp1

DISTRIBUTIVE MECHANISM, MINIMAL CTD SUBSTRATE, AND ACTIVE SITE MAPPING^*

Stéphane Hausmann, Hediye Erdjument-Bromage, and Stewart Shuman

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

Schizosaccharomyces pombe Fcp1 is an essential protein serine phosphatase that preferentially dephosphorylates Ser² of the RNA polymerase II C-terminal domain (CTD) heptad repeat Y¹S²P³T⁴S⁵P⁶S⁷. 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 S⁵P⁶S⁷Y¹S²P³T⁴; and (iii) single alanine mutations of flanking residues Tyr¹ or Pro³ 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 Fcp1 orthologs and likely comprise the active site.

Received for publication, November 14, 2003 , and in revised form, December 28, 2003.

^* This work was supported by National Institutes of Health Grant GM52470. The Microchemistry Core Laboratory is supported by NCI Cancer Center Support Grant P30 CA08748. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 212-639-7145; Fax: 212-717-3623; E-mail: s-shuman{at}ski.mskcc.org.

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