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Originally published In Press as doi:10.1074/jbc.M100980200 on February 13, 2001

J. Biol. Chem., Vol. 276, Issue 20, 17261-17266, May 18, 2001
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Structure-Function Analysis of the Active Site Tunnel of Yeast RNA Triphosphatase*

Martin Bisaillon and Stewart ShumanDagger

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

Cet1, the RNA triphosphatase component of the yeast mRNA capping apparatus, catalyzes metal-dependent gamma  phosphate hydrolysis within the hydrophilic interior of a topologically closed 8-strand beta  barrel (the "triphosphate tunnel"). We used structure-guided alanine scanning to identify 6 side chains within the triphosphate tunnel that are essential for phosphohydrolase activity in vitro and in vivo: Arg393, Glu433, Arg458, Arg469, Asp471 and Thr473. Alanine substitutions at two positions, Asp377 and Lys409, resulted in partial catalytic defects and a thermosensitive growth phenotype. Structure-function relationships were clarified by introducing conservative substitutions. Five residues were found to be nonessential: Lys309, Ser395, Asp397, Lys427, Asn431, and Lys474. The present findings, together with earlier mutational analyses, reveal an unusually complex active site in which 15 individual side chains in the tunnel cavity are important for catalysis, and each of the 8 strands of the beta  barrel contributes at least one functional constituent. The active site residues fall into three classes: (i) those that participate directly in catalysis via coordination of the gamma  phosphate or the metal; (ii) those that make critical water-mediated contacts with the gamma  phosphate or the metal; and (iii) those that function indirectly via interactions with other essential side chains or by stabilization of the tunnel structure.

* This research was supported in part by National Institutes of Health Grant GM52470 (to S. S.) and a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada (to M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Fax: 212-717-3623; E-mail: s-shuman@ski.mskcc.org.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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