Journal article
Identification of overlapping but distinct cAMP and cGMP interaction sites with cyclic nucleotide phosphodiesterase 3A by site-directed mutagenesis and molecular modeling based on crystalline PDE4B
Protein science, v 10(8), pp 1481-1489
01 Aug 2001
PMID: 11468344
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
Cyclic nucleotide phosphodiesterase 3A (PDE3A) hydrolyzes cAMP to AMP, but is competitively inhibited by cGMP due to a low k
cat
despite a tight K
m
. Cyclic AMP elevation is known to inhibit all pathways of platelet activation, and thus regulation of PDE3 activity is significant. Although cGMP elevation will inhibit platelet function, the major action of cGMP in platelets is to elevate cAMP by inhibiting PDE3A. To investigate the molecular details of how cGMP, a similar but not identical molecule to cAMP, behaves as an inhibitor of PDE3A, we constructed a molecular model of the catalytic domain of PDE3A based on homology to the recently determined X-ray crystal structure of PDE4B. Based on the excellent fit of this model structure, we mutated nine amino acids in the putative catalytic cleft of PDE3A to alanine using site-directed mutagenesis. Six of the nine mutants (Y751A, H840A, D950A, F972A, Q975A, and F1004A) significantly decreased catalytic efficiency, and had k
cat
/K
m
less than 10% of the wild-type PDE3A using cAMP as substrate. Mutants N845A, F972A, and F1004A showed a 3- to 12-fold increase of K
m
for cAMP. Four mutants (Y751A, H840A, D950A, and F1004A) had a 9- to 200-fold increase of K
i
for cGMP in comparison to the wild-type PDE3A. Studies of these mutants and our previous study identified two groups of amino acids: E866 and F1004 contribute commonly to both cAMP and cGMP interactions while N845, E971, and F972 residues are unique for cAMP and the residues Y751, H836, H840, and D950 interact with cGMP. Therefore, our results provide biochemical evidence that cGMP interacts with the active site residues differently from cAMP.
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Details
- Title
- Identification of overlapping but distinct cAMP and cGMP interaction sites with cyclic nucleotide phosphodiesterase 3A by site-directed mutagenesis and molecular modeling based on crystalline PDE4B
- Creators
- Wei Zhang - Temple UniversityHengming Ke - University of North Carolina at Chapel HillAnna P. Tretiakova - Hahnemann University HospitalBradford Jameson - Hahnemann University HospitalRobert W. Colman - Temple University
- Publication Details
- Protein science, v 10(8), pp 1481-1489
- Publisher
- Cold Spring Harbor Laboratory Press
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- [Retired Faculty]
- Web of Science ID
- WOS:000169984100001
- Scopus ID
- 2-s2.0-0034924752
- Other Identifier
- 991019168679404721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemistry & Molecular Biology