Abstract
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 EC(50)(Ca(2+)) 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 EC(50)(Ca(2+)) 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 Ca(2+) concentration of 50-100 nm.
Highlights
The nitric-oxide synthases catalyze formation of NO and L-citrulline from L-arginine and O2, with NADPH as the electron donor (1)
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
Biochemical Implications—The effects of the S1179D substitution on EC50(Ca2ϩ) values that we report here are similar to those produced by an S617D substitution (21)
Summary
Expression and Purification of Proteins—The pCWori vector (22, 23) used for bacterial expression of tagged eNOS has been described in detail previously (21). Final eNOS concentrations in dialysates were determined from optical absorbance spectra as described previously (21) Enzyme prepared in this manner was used in CaM binding and enzyme activity assays. Calibration of the indicators in this manner allows the relationships between CaM binding, enzyme activation and [Ca2ϩ] to be quantitatively related according to a previously described sequential model in which formation of the CaM-eNOS complex requires Ca2ϩ binding to one EF hand pair in CaM, and activation requires binding to both EF hand pairs (21). Enzyme activation versus [Ca2ϩ] for native or mutant enzyme were simultaneously fitted to Equations 2 and 3 using the Excel solver to derive the fits shown in Fig. 4, with the K1K2 and K3K4 values listed in Table
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
