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J. Biol. Chem., Vol. 276, Issue 18, 14804-14813, May 4, 2001
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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.
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*
§,
*
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.
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.
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