Journal article
Probing Domain Functions of Chimeric PDE6α′/PDE5 cGMP-Phosphodiesterase
The Journal of biological chemistry, Vol.273(38), pp.24485-24490
09/18/1998
DOI: 10.1074/jbc.273.38.24485
PMID: 9733741
Abstract
Chimeric cGMP phosphodiesterases (PDEs) have been constructed using components of the cGMP-binding PDE (PDE5) and cone photoreceptor phosphodiesterase (PDE6α′) in order to study structure and function of the photoreceptor enzyme. A fully functional chimeric PDE6α′/PDE5 enzyme containing the PDE6α′ noncatalytic cGMP-binding sites, and the PDE5 catalytic domain has been efficiently expressed in the baculovirus/High Five cell system. The catalytic properties of this chimera were practically indistinguishable from those of PDE5, whereas the noncatalytic cGMP binding was similar to that of native purified PDE6α′. The inhibitory γ subunit of PDE6 (Pγ) enhanced the affinity of cGMP binding at noncatalytic sites of native PDE6α′ by ∼6-fold. The polycationic region of Pγ, Pγ-24–45, was mainly responsible for this effect, while the inhibitory domain of Pγ, Pγ-63–87, was ineffective. On the contrary, Pγ failed to inhibit catalytic activity of the chimeric PDE6α′/PDE5 or to modulate its noncatalytic cGMP binding.
Substitutions of Ala residues for the conserved Asn, Asn193 or Asn402, in the two N(K/R)XD-like motifs of the chimeric PDE noncatalytic cGMP-binding sites, each led to a loss of the noncatalytic cGMP binding. Our data suggest that both putative noncatalytic sites of PDE6α′ are important for binding of cGMP, and that the two binding sites are coupled. Furthermore, mutation Asn402 → Ala resulted in an approximately 10-fold increase of theK m value for cGMP, indicating that occupation of the noncatalytic cGMP- binding sites of PDE6α′ may regulate catalytic properties of the enzyme.
Details
- Title: Subtitle
- Probing Domain Functions of Chimeric PDE6α′/PDE5 cGMP-Phosphodiesterase
- Creators
- Alexey E GranovskyMichael NatochinRandall L McEntafferTamara L HaikSharron H FrancisJackie D CorbinNikolai O Artemyev
- Resource Type
- Journal article
- Publication Details
- The Journal of biological chemistry, Vol.273(38), pp.24485-24490
- DOI
- 10.1074/jbc.273.38.24485
- PMID
- 9733741
- NLM abbreviation
- J Biol Chem
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- Language
- English
- Date published
- 09/18/1998
- Academic Unit
- Molecular Physiology and Biophysics; Iowa Neuroscience Institute; Physics and Astronomy; Ophthalmology and Visual Sciences
- Record Identifier
- 9984025668702771
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