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

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP; EC ) 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 iden...

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Bibliographic Details
Published in:The Journal of biological chemistry 2001-05, Vol.276 (18), p.14804-14813
Main Authors: Lee, J, Gravel, M, Gao, E, O'Neill, R C, Braun, P E
Format: Article
Language:English
Subjects:
2',3'-Cyclic Nucleotide 3'-Phosphodiesterase
2',3'-Cyclic-Nucleotide Phosphodiesterases - antagonists & inhibitors
2',3'-Cyclic-Nucleotide Phosphodiesterases - chemistry
2',3'-Cyclic-Nucleotide Phosphodiesterases - genetics
2',3'-Cyclic-Nucleotide Phosphodiesterases - metabolism
5,5'-Dithiobis-(2-nitrobenzoic acid)
Amino Acid Sequence
Animals
Base Sequence
Catalytic Domain
Cysteine - metabolism
Dithionitrobenzoic Acid - pharmacology
DNA Primers
Enzyme Inhibitors - pharmacology
Histidine - metabolism
Kinetics
Molecular Sequence Data
Mutagenesis, Site-Directed
Phosphoric Diester Hydrolases
Rats
Recombinant Proteins - antagonists & inhibitors
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Sequence Homology, Amino Acid
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Description
Summary:2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP; EC ) 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 k(cat) values 1000-fold lower than wild-type CNP-CF, but K(m) 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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M009434200