Phosphorylation within an autoinhibitory domain in endothelial nitric oxide synthase reduces the Ca2+ 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 Call dependencies for calmodulin binding and enzyme activation. A phosphomimetic S617D substitution doubles the maximum calmodulin-dependent enzyme activity and decreases the EC50(Ca2+) 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 EC50(Ca2+) 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 EC50(Ca2+) 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 EC50(Ca2+) values and maximum calmodulin-dependent enzyme activity are predicted to contribute equally to phosphorylationdependent enhancement of NO production during a typical agonist-evoked Ca2+ transient, while the reduction in EC50(Ca2+) values is predicted to be the major contributor to enhancement at resting free Call concentrations.