title: Structural elements contribute to the calcium/calmodulin dependence on enzyme activation in human endothelial nitric-oxide synthase.
abstract: Two regions, located at residues 594-606/614-645 and residues 1165-1178, are present in the reductase domain of human endothelial nitric-oxide synthase (eNOS) but absent in its counterpart, inducible nitric-oxide synthase (iNOS). We previously demonstrated that removing residues 594-606/614-645 resulted in an enzyme (Delta45) containing an intrinsic calmodulin (CaM) purified from an Sf9/baculovirus expression system (Chen, P.-F., and Wu, K.K. (2000) J. Biol. Chem. 275, 13155-13163). Here we have further elucidated the differential requirement of Ca2+/CaM for enzyme activation between eNOS and iNOS by either deletion of residues 1165-1178 (Delta14) or combined deletions of residues 594-606/614-645 and 1165-1178 (Delta45/ Delta14) from eNOS to mimic iNOS. We measured the catalytic rates using purified proteins completely free of CaM. Steady-state analysis indicated that the Delta45 supported NO synthesis in the absence of CaM at 60% of the rate in its presence, consistent with our prior result that CaM-bound Delta45 retained 60% of its activity in the presence of 10 mm EGTA. Mutant Delta14 displayed a 1.5-fold reduction of EC50 for Ca2+/CaM-dependence in l-citrulline formation, and a 2-4-fold increase in the rates of NO synthesis, NADPH oxidation, and cytochrome c reduction relative to the wild type. The basal rates of double mutant Delta45/Delta14 in NO production, NADPH oxidation, and cytochrome c reduction were 3-fold greater than those of CaM-stimulated wild-type eNOS. Interestingly, all three activities of Delta45/ Delta14 were suppressed rather than enhanced by Ca2+/CaM, indicating a complete Ca2+/CaM independence for those reactions. The results suggest that the Ca2+/CaM-dependent catalytic activity of eNOS appears to be conferred mainly by these two structural elements, and the interdomain electron transfer from reductase to oxygenase domain does not require Ca2+/CaM when eNOS lacks these two segments.
authors.affiliation: Vascular Biology Research Center and Division of Hematology, Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, Texas 77225, USA. pei-feng.chen@uth.tmc.edu
authors.forename: Pei-Feng
authors.initials: PF
authors.lastname: Chen
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002614:Chelating Agents / Q000494:pharmacology; D002956:Citrulline / Q000737:chemistry; D003470:Culture Media / Q000494:pharmacology; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005292:Ferricyanides / Q000737:chemistry; D017353:Gene Deletion; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D009154:Mutation; D009249:NADP / Q000737:chemistry; D009566:Nitrates / Q000737:chemistry; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D009573:Nitrites / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D002147:Calmodulin; D002614:Chelating Agents; D003470:Culture Media; D018076:DNA, Complementary; D005292:Ferricyanides; D009566:Nitrates; D009573:Nitrites; C007931:hexacyanoferrate III; D002956:Citrulline; D009569:Nitric Oxide; D009249:NADP; D045304:Cytochromes c; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen; D002118:Calcium