title: Characterization of the roles of the 594-645 region in human endothelial nitric-oxide synthase in regulating calmodulin binding and electron transfer.
abstract: It has been postulated that a segment (residues 594-645) inserted in the FMN subdomain of human endothelial nitric-oxide synthase (eNOS) plays a crucial role in controlling Ca(2+)-dependent CaM binding for eNOS activity. To investigate its functions, we expressed human eNOS in a baculovirus system with deletion of a 45-residue segment from this region (residues 594-606 and 614-645, designated as Delta45eNOS), and characterized the purified mutant enzyme. In contrast with wild-type eNOS, Delta45eNOS exhibited characteristics resembling inducible NOS (iNOS). It contained an endogenously bound CaM, which was essential in folding and stabilizing this mutant enzyme, and retained 60% of L-citrulline formation in 5 mM EGTA. We also produced four N-terminally truncated reductase domains with or without the 45-residue segment, and either including or excluding the CaM-binding sequence. Basal cytochrome c reductase activity of reductase domains without the 45-residue segment was up to 20 fold greater than that of corresponding insert-containing domains, and higher than CaM-stimulated activity of the wild-type enzyme. A series of mutants with smaller fragment deletion in this region such as Delta594-604, Delta605-612, Delta613-625, Delta626-634, Delta632-639, and Delta640-645 mutants were further characterized. The crude lysate of mutants Delta613-625 and Delta632-639 did not show activity in the presence of Ca(2+)/CaM, while other four mutants had activity comparable to that of WTeNOS. The purified Delta594-604 and Delta605-612 proteins had a 3-5-fold higher affinity for Ca(2+)/CaM, but their L-citrulline forming activity was still 80% dependent upon the addition of Ca(2+)/CaM. Both mutants exhibited a low level of the cytochrome c and ferricyanide reductase activities, which either did not respond to (Delta594-604) or slightly enhanced by (Delta605-612) the exogenous CaM. In contrast, activities of Delta626-634 and Delta640-645 like those of WTeNOS were largely Ca(2+)/CaM-dependent. Thus, our findings indicate that the N-terminal half of the 594-645 segment containing residues 594-612 plays a significant role in regulating Ca(2+)/CaM binding.
authors.affiliation: Vascular Biology Research Center and Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas 77225, USA. pfchen@heart.med.uth.tmc.edu
authors.forename: P F
authors.initials: PF
authors.lastname: Chen
authors.forename: K K
authors.initials: KK
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D002956:Citrulline / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007313:Insecta; D007527:Isoenzymes; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009245:NADH Dehydrogenase / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D009573:Nitrites / Q000378:metabolism; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
chemical_list: D002147:Calmodulin; D007527:Isoenzymes; D009566:Nitrates; D009573:Nitrites; D002956:Citrulline; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D009245:NADH Dehydrogenase; D002118:Calcium