期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 277, 期 49, 页码 47581-47587出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M206088200
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资金
- NHLBI NIH HHS [HL46494] Funding Source: Medline
- NIDDK NIH HHS [DK58277, DK40029] Funding Source: Medline
Substrate binding to the phosphodiesterase-5 (PDE5) catalytic site increases cGMP binding to the regulatory domain (R domain). The latter promotes PDE5 phosphorylation by cyclic nucleotide-dependent protein kinases, which activates catalysis, enhances allosteric cGMP binding, and causes PDE5A1 to apparently elongate. A human PDE5A1 R domain fragment (Val(46)-Glu(539)) containing the phosphorylation site (Ser(102)) and allosteric cGMP-binding sites was studied. The rate, cGMP dependence, and stoichiometry of phosphorylation of the PDE5 R domain by the catalytic subunit of cAMP-dependent protein kinase are comparable with that of the holoenzyme. Migration in native polyacrylamide gels suggests that either cGMP binding or phosphorylation produces distinct conformers of the R domain. Phosphorylation of the R domain increases affinity for cGMP similar to10-fold (K-D values 97.8+/-17 and 10.0+/-0.5 nM for unphospho- and phospho-R domains, respectively). [3 H]cGMP dissociates from the phospho-R domain with a single rate (t(1/2)=339+/-30 min) compared with the biphasic pattern of the unphospho-R domain (t(1/2)=39.0+/-4.8 and 265+/-28 min, for the fast and slow components, respectively). Thus, cGMP-directed regulation of PDE5 phosphorylation and the resulting increase in cGMP binding affinity occur largely within the R domain. Conformational change(s) elicited by phosphorylation of the R domain within the PDE5 holoenzyme may also cause or participate in stimulating catalysis.
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