Abstract
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 Ser1179 occurs 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 Ser1179 phosphorylation 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 Ser1179 phosphorylation regulates eNOS and provides a foundation to compare its regulation by other phosphorylation events.
Highlights
Enzyme Species Distribution and FMNhq Reactivity toward Cytochrome c—The simulations can predict how eNOSr would distribute among the four species involved in its catalytic cycle (Fig. 1; FMNsq open, FMNsq closed, FMNhq closed, and FMNhq open) during its reaction with excess cytochrome c and can predict how CaM binding or the S1179D substitution will influence the distribution
As explained previously [16], the analysis first generates combinations of allowable conformational switching and interflavin ET rate pairs that each support the experimentally observed rate of electron flux to cytochrome c
Faster conformational switching rates can be paired with slower rates of interflavin ET and vice versa to support the observed steady-state rate of electron flux through each eNOSr protein to cytochrome c
Summary
Enzyme Species Distribution and FMNhq Reactivity toward Cytochrome c—The simulations can predict how eNOSr would distribute among the four species involved in its catalytic cycle (Fig. 1; FMNsq open, FMNsq closed, FMNhq closed, and FMNhq open) during its reaction with excess cytochrome c and can predict how CaM binding or the S1179D substitution will influence the distribution. This creates an equivalent population of reacted, conformationally open molecule
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