Phosphorylation Controls Endothelial Nitric-oxide Synthase by Regulating Its Conformational Dynamics.
The activity of endothelial NO synthase (eNOS) is triggered by calmodulin (CaM) binding and is often further regulated by phosphorylation at several positions in the enzyme. Phosphorylation at Ser1179occurs in response to diverse physiologic stimuli and increases the NO synthesis and cytochrome c reductase activities of eNOS, thereby enhancing its participation in biological signal cascades. Despite its importance, the mechanism by which Ser1179phosphorylation increases eNOS activity is not understood. To address this, we used stopped-flow spectroscopy and computer modeling approaches to determine how the phosphomimetic mutation (S1179D) may impact electron flux through eNOS and the conformational behaviors of its reductase domain, both in the absence and presence of bound CaM. We found that S1179D substitution in CaM-free eNOS had multiple effects; it increased the rate of flavin reduction, altered the conformational equilibrium of the reductase domain, and increased the rate of its conformational transitions. We found these changes were equivalent in degree to those caused by CaM binding to wild-type eNOS, and the S1179D substitution together with CaM binding caused even greater changes in these parameters. The modeling indicated that the changes caused by the S1179D substitution, despite being restricted to the reductase domain, are sufficient to explain the stimulation of both the cytochrome c reductase and NO synthase activities of eNOS. This helps clarify how Ser1179phosphorylation regulates eNOS and provides a foundation to compare its regulation by other phosphorylation events.
From the Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195 haquem@ccf.org.
Mohammad Mahfuzul
MM
Haque
From the Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195.
Sougata Sinha
SS
Ray
From the Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195 stuehrd@ccf.org.
Dennis J
DJ
Stuehr
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D045304:Cytochromes c / Q000378:metabolism; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation
D005415:Flavins; D009569:Nitric Oxide; D045304:Cytochromes c; D052250:Nitric Oxide Synthase Type III
catalysis; eNOS; electron transfer; fast reaction kinetics; flavoprotein; heme reduction; kinetic model; nitric oxide synthase; pre-steady-state kinetics; simulation