Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation

Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the n egatively charged aspartate (S1179D eNOS) increases nitric oxide (NO) produ ction constitutively, in the absence of agonist challenge. Here, me examine the potential mechanism of how aspartate at 1179 increases eNOS activity u sing purified proteins. Examination of NO production and cytochrome c reduc tion resulted in no substantial changes in the K-m/EC50 for L-arginine, cal modulin, and calcium, whereas there was a a-fold increase in the rate of NO production for S1179D and a 2-4 fold increase in reductase activity (based on cytochrome c reduction). The observed increase in activity for both ass ays of NOS function indicates that a faster rate of electron flux through t he reductase domain is likely the rate-limiting step in NO formation from e NOS, In addition, S1179D eNOS did show an increased resistance to inactivat ion by EGTA compared with mild type eNOS. These results suggest that a nega tive charge imposed at serine 1179, either by phosphorylation or by replace ment with aspartate, increases eNOS catalytic activity by increasing electr on flux at the reductase domain and by reducing calmodulin dissociation fro m activated eNOS when calcium levels are low.