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 ⌬45eNOS), and characterized the purified mutant enzyme. In contrast with wild-type eNOS, ⌬45eNOS 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 ⌬594 -604, ⌬605-612, ⌬613-625, ⌬626 -634, ⌬632-639, and ⌬640 -645 mutants were further characterized. The crude lysate of mutants ⌬613-625 and ⌬632-639 did not show activity in the presence of Ca 2؉ /CaM, while other four mutants had activity comparable to that of WTeNOS. The purified ⌬594 -604 and ⌬605-612 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 (⌬594 -604) or slightly enhanced by (⌬605-612) the exogenous CaM. In contrast, activities of ⌬626 -634 and ⌬640 -645 like those of WTeNOS were largely Ca 2؉ /CaMdependent. 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.Nitric oxide ( ⅐ NO) is an important signal mediator in diverse physiological and pathological events of mammals (1-3). This molecule is coproduced with L-citrulline from L-arginine by three different nitric-oxide synthase (NOS) 1 isoforms, all of which share similar biochemical composition and enzymatic characteristics, and possess a bidomain structure (4 -8). The C-terminal half (reductase domain) contains the NADPH-, FAD-, and FMN-binding sites and the N-terminal half (oxygenase domain) contains the heme-, BH 4 -, and L-arginine-binding sites (9 -11). These two domains are connected by a calmodulinbinding region (6,8).Despite structural similarities, the NOS isoforms can be grouped into two distinct classes according to their mode of expression and dependence on Ca 2ϩ . Constitutive NOS (cNOS) isoforms including endothelial NOS (eNOS) and neuronal NOS (nNOS), are constitutively expressed as latent enzymes that require a higher conce...

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

“…A putative 'auto-inhibitory element', a -50 amino acid residue segment present in the FMN-binding domain of eNOS impedes CaM binding to the enzyme, and thus high Ca 2+ level is required for the enzyme activation (Nishida and Ortiz de Montellano, 1999;Chen and Wu, 2000). While Ca 2+ is a major regulator of eNOS activity, it has become evident that eNOS activity is also regulated by other mechanisms such as protein-protein interactions and post-translational modifications.…”

Shear stress stimulates phosphorylation of eNOS at Ser635by a protein kinase A-dependent mechanism

“…It has been recognized that a ϳ50-residue segment present in the flavin mononucleotide -binding domain of eNOS (residue 596-647 based on the bovine sequence) represents a putative "autoinhibitory element" that impedes CaM binding to eNOS and electron flux between the two monomers (7,38). Deletion of this element from eNOS decreases Ca 2ϩ and CaM requirement for enzyme activation and enhances maximal activity (7,38).…”

“…Deletion of this element from eNOS decreases Ca 2ϩ and CaM requirement for enzyme activation and enhances maximal activity (7,38). Because Ser 635 is located in this "autoinhibitory element," it is a reasonable speculation that phosphorylation of Ser 635 may affect CaM binding and electron transfer of the enzyme.…”

Identification of Caveolin-1-interacting Sites in Neuronal Nitric-oxide Synthase

“…Deletion of either element affects the Ca 2ϩ /CaM sensitivity of nNOS and eNOS and allows electron transfers to proceed even in the absence of CaM (11)(12)(13)(14)(15)(16)(17). To investigate how caveolin binding regulates these CaM-independent electron transfers, we examined two deletion mutants of nNOS: ⌬40, in which 40 amino acids have been deleted from the FMN binding subdomain and ⌬C33, in which 33 amino acids have been truncated from the C-terminal end (Fig.…”

“…Peptides derived from this putative autoinhibitory domain in eNOS inhibited CaM binding and Ca 2ϩ activation of both nNOS and eNOS (11). Deletion of the autoinhibitory domains of eNOS and nNOS activated the reductase domains in the absence of CaM and affected Ca 2ϩ /CaM-dependent NO formation (12)(13)(14)(15)(16). A mutant nNOS in which this region was deleted (⌬40) was 10% as active as the wild-type enzyme in the absence of Ca 2ϩ /CaM (16).…”