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Originally published In Press as doi:10.1074/jbc.M009434200 on February 5, 2001

J. Biol. Chem., Vol. 276, Issue 18, 14804-14813, May 4, 2001
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Identification of Essential Residues in 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase
CHEMICAL MODIFICATION AND SITE-DIRECTED MUTAGENESIS TO INVESTIGATE THE ROLE OF CYSTEINE AND HISTIDINE RESIDUES IN ENZYMATIC ACTIVITY*

John LeeDagger §, Michel Gravel§, Enoch Gao, Ryan C. O'Neill, and Peter E. Braun

From the Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP; EC 3.1.4.37) catalyzes in vitro hydrolysis of 3'-phosphodiester bonds in 2',3'-cyclic nucleotides to produce 2'-nucleotides exclusively. N-terminal deletion mapping of the C-terminal two-thirds of recombinant rat CNP1 identified a region that possesses the catalytic domain, with further truncations abolishing activity. Proteolysis and kinetic analysis indicated that this domain forms a compact globular structure and contains all of the catalytically essential features. Subsequently, this catalytic fragment of CNP1 (CNP-CF) was used for chemical modification studies to identify amino acid residues essential for activity. 5,5'-Dithiobis-(2-nitrobenzoic acid) modification studies and kinetic analysis of cysteine CNP-CF mutants revealed the nonessential role of cysteines for enzymatic activity. On the other hand, modification studies with diethyl pyrocarbonate indicated that two histidines are essential for CNPase activity. Consequently, the only two conserved histidines, His-230 and His-309, were mutated to phenylalanine and leucine. All four histidine mutants had kcat values 1000-fold lower than wild-type CNP-CF, but Km values were similar. Circular dichroism studies demonstrated that the low catalytic activities of the histidine mutants were not due to gross changes in secondary structure. Taken together, these results demonstrate that both histidines assume critical roles for catalysis.

* This work was supported by a grant from the Medical Research Council of Canada (MRC).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 Recipient of a studentship from the MRC and the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.

§ These authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-7281; Fax: 514-398-7384; E-mail: braun@med.mcgill.ca.

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