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Effects of Combined Phosphorylation at Ser-617 and Ser-1179 in Endothelial Nitric-oxide Synthase on EC50(Ca2+) Values for Calmodulin Binding and Enzyme Activation*

By Quang-Kim Tran, Jared Leonard, D. J. Black, Owen W. Nadeau, Igor G. Boulatnikov and Anthony Persechini


We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist- or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC50(Ca2+) values for calmodulin binding and enzyme activation from the control values of 182 ± 2 and 422 ± 22 nm to 116 ± 2 and 300 ± 10 nm. These are similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q. K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry 47, 7557–7566). Although combining substitutions at Ser-617 and Ser-1179 has no additional effect on maximal synthase activity, cooperativity between the two substitutions completely disinhibits reductase activity and further reduces the EC50(Ca2+) values for calmodulin binding and enzyme activation to 77 ± 2 and 130 ± 5 nm. We have confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 and phosphomimetic substitutions at these positions have similar functional effects. Changes in the biochemical properties of eNOS produced by combined phosphorylation at Ser-617 and Ser-1179 are predicted to substantially increase synthase activity in cells at a typical basal free Ca2+ concentration of 50–100 nm

Topics: Enzyme Catalysis and Regulation
Publisher: American Society for Biochemistry and Molecular Biology
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Provided by: PubMed Central
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