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 (⌬45) 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 Ca 2؉ / CaM for enzyme activation between eNOS and iNOS by either deletion of residues 1165-1178 (⌬14) or combined deletions of residues 594 -606/614 -645 and 1165-1178 (⌬45/ ⌬14) from eNOS to mimic iNOS. We measured the catalytic rates using purified proteins completely free of CaM. Steady-state analysis indicated that the ⌬45 supported NO synthesis in the absence of CaM at 60% of the rate in its presence, consistent with our prior result that CaMbound ⌬45 retained 60% of its activity in the presence of 10 mM EGTA. Mutant ⌬14 displayed a 1.5-fold reduction of EC 50 for Ca 2؉ /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 ⌬45/⌬14 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 ⌬45/ ⌬14 were suppressed rather than enhanced by Ca /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 Ca 2؉ /CaM when eNOS lacks these two segments. Nitric-oxide synthase (NOS)1 catalyzes the synthesis of NO through a series of electron transfers from the C-terminal reductase domain, which harbors the FAD and FMN cofactors and the NADPH binding site, to the N-terminal oxygenase domain, which contains the heme catalytic center, the H 4 B cofactor, and the arginine binding sites (1-8). An electron generated by NADPH is transferred in tandem to FAD and FMN and then to the heme, which has been proposed to be facilitated by CaM bound to a site situated between these two domains (9). The NOS family comprises three isoforms that share domain structures, sequence homology, and catalytic properties (10 -12). Despite these similarities, there are considerable differences among the NOS isoforms with respect to their cellular expressions, Ca 2ϩ -dependent CaM activation, and rate of electron transfer. Two isoforms, i.e. nNOS and eNOS, are constitutively expressed NOSs (cNOSs), but their expressed enzymes are latent until CaM binding is elicited by an elevated intracellular calcium level (13). In contrast, iNOS is absent or expressed in low abundance at the resting state, and its expression is induced by cytokines and endotoxins. The expressed iNOS is catalytically active, thought to be due to its hi...

Phosphorylation of Endothelial Nitric-oxide Synthase Regulates Superoxide Generation from the Enzyme

“…This indicates that phosphorylation of eNOS increases the association between eNOS and caveolin. As such, our in vitro data are consistent with the hypothesis that Thr-495 phosphorylation negatively regulates eNOS activity (13,20,22,44,52,55,56). Previous reports have indicated that phosphorylation of eNOS at Ser-1177 leads to a dissociation of eNOS and caveolin; however, this dissociation was dependent upon the induction of caveolin-dependent endocytosis (57,58).…”

Surface Charge Interactions of the FMN Module Govern Catalysis by Nitric-oxide Synthase

“…This is consistent with the autoinhibitory insert and C-terminal regulatory elements being required to block heme reduction in CaM-free NOS (26,35,40). Regarding heme reduction in the CaM-bound mutants, Glu 762 is clearly the most important of the six acidic residues because Arg and Ala substitutions at this position slowed down nNOS heme reduction by 95% and caused corresponding decreases in NO synthesis activity.…”

Recruitment of governing elements for electron transfer in the nitric oxide synthase family

“…The structural comparisons shown in the present report also confirm that these residues have the potential to confer additional functional control on the NOS isoforms. This was demonstrated amply by the work of Roman and colleagues (24,25) prior to any structural information and was confirmed and extended by other laboratories (29,43).…”

“…Also, the ability, particularly in eNOS, to control the production of NO through phosphorylation of Ser-1197 in the C-terminal tail (26,27,42) is extremely important for function. Recent reports have addressed the consequences of the truncation of the C-terminal sequences of NOS on the various activities of the enzyme (24,25,29,43).…”