Phosphorylation within an autoinhibitory domain in endothelial nitric oxide synthase reduces the Ca(2+) concentrations required for calmodulin to bind and activate the enzyme.
We have investigated the effects of phosphorylation at Ser-617 and Ser-635 within an autoinhibitory domain (residues 595-639) in bovine endothelial nitric oxide synthase on enzyme activity and the Ca (2+) dependencies for calmodulin binding and enzyme activation. A phosphomimetic S617D substitution doubles the maximum calmodulin-dependent enzyme activity and decreases the EC 50(Ca (2+)) values for calmodulin binding and enzyme activation from the wild-type values of 180 +/- 2 and 397 +/- 23 nM to values of 109 +/- 2 and 258 +/- 11 nM, respectively. Deletion of the autoinhibitory domain also doubles the maximum calmodulin-dependent enzyme activity and decreases the EC 50(Ca (2+)) values for calmodulin binding and calmodulin-dependent enzyme activation to 65 +/- 4 and 118 +/- 4 nM, respectively. An S635D substitution has little or no effect on enzyme activity or EC 50(Ca (2+)) values, either alone or when combined with the S617D substitution. These results suggest that phosphorylation at Ser-617 partially reverses suppression by the autoinhibitory domain. Associated effects on the EC 50(Ca (2+)) values and maximum calmodulin-dependent enzyme activity are predicted to contribute equally to phosphorylation-dependent enhancement of NO production during a typical agonist-evoked Ca (2+) transient, while the reduction in EC 50(Ca (2+)) values is predicted to be the major contributor to enhancement at resting free Ca (2+) concentrations.
Division of Molecular Biology and Biochemistry, University of Missouri, Kansas City, Missouri 64110-2499, USA.
Quang-Kim
QK
Tran
Jared
J
Leonard
D J
DJ
Black
Anthony
A
Persechini
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers; D007700:Kinetics; D016296:Mutagenesis; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D002147:Calmodulin; D017931:DNA Primers; D010750:Phosphoproteins; D011994:Recombinant Proteins; D052250:Nitric Oxide Synthase Type III; D002118:Calcium