| Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity. | |
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MedLine Citation:
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PMID: 1662209 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels. |
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Authors:
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J B Shabb; B D Buzzeo; L Ng; J D Corbin |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: The Journal of biological chemistry Volume: 266 ISSN: 0021-9258 ISO Abbreviation: J. Biol. Chem. Publication Date: 1991 Dec |
Date Detail:
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Created Date: 1992-02-07 Completed Date: 1992-02-07 Revised Date: 2009-11-19 |
Medline Journal Info:
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Nlm Unique ID: 2985121R Medline TA: J Biol Chem Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 24320-6 Citation Subset: IM |
Affiliation:
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Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Animals Base Sequence Binding Sites / genetics Cattle Cyclic AMP / metabolism* Cyclic GMP / metabolism* Enzyme Activation Molecular Sequence Data Mutation* Protein Kinases / genetics*, metabolism Sequence Alignment Sequence Homology, Nucleic Acid Substrate Specificity / genetics |
| Grant Support | |
ID/Acronym/Agency:
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DK40029/DK/NIDDK NIH HHS; T35DK07383/DK/NIDDK NIH HHS |
| Chemical | |
Reg. No./Substance:
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60-92-4/Cyclic AMP; 7665-99-8/Cyclic GMP; EC 2.7.-/Protein Kinases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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