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 negatively charged aspartate (S1179D eNOS) increases nitric oxide (NO) production constitutively, in the absence of agonist challenge. Here, we examine the potential mechanism of how aspartate at 1179 increases eNOS activity using purified proteins. Examination of NO production and cytochrome c reduction resulted in no substantial changes in the K(m)/EC(50) for L-arginine, calmodulin, and calcium, whereas there was a 2-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 assays of NOS function indicates that a faster rate of electron flux through the reductase domain is likely the rate-limiting step in NO formation from eNOS. In addition, S1179D eNOS did show an increased resistance to inactivation by EGTA compared with wild type eNOS. These results suggest that a negative charge imposed at serine 1179, either by phosphorylation or by replacement with aspartate, increases eNOS catalytic activity by increasing electron flux at the reductase domain and by reducing calmodulin dissociation from activated eNOS when calcium levels are low.
McCabe
T J
TJ
Department of Pharmacology and Molecular Cardiobiology Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Fulton
D
D
Roman
L J
LJ
Sessa
W C
WC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D019281:Dimerization; D004533:Egtazic Acid / Q000494:pharmacology; D004583:Electrons; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D009154:Mutation; D009245:NADH Dehydrogenase / Q000378:metabolism; D009249:NADP / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D055672:Static Electricity
D002147:Calmodulin; D011518:Proto-Oncogene Proteins; D009569:Nitric Oxide; D004533:Egtazic Acid; D009249:NADP; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D009245:NADH Dehydrogenase; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D002118:Calcium