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.

No abstract

The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain ofE. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds N'0-nitroarginine dependent on the presence of bound tetrahydrobiopterin and exhibits a Kd of 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca 2؉ -dependent (cNOS) or Ca 2؉ -independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [ 3 H]N G -nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.Nitric oxide is a ubiquitous cell-signaling molecule, with protean roles in physiology and pathophysiology (1-3). Encoded by distinct genes, mammalian NO synthases (NOSs) 1 comprise a family of three calmodulin-dependent biopterohemoflavoproteins that are functionally distinguished by their modes of regulation (4). The two constitutively expressed isoforms of NOS (cNOSs), first identified in neuronal cells (nNOS) and endothelial cells (eNOS), remain dormant until calcium/calmodulin (Ca 2ϩ /CaM) binding is actuated by transient elevations in intracellular Ca 2ϩ . This Ca 2ϩ -dependent mode of regulation provides pulses of NO for moment-to-moment modulation of vascular tone and neurosignaling. In contrast, activity of the immunostimulant-induced isoform of NOS (iNOS) is Ca 2ϩ -independent, providing continuous high output NO generation for host defense. A remarkably high affinity for CaM, even at basally low levels of intracellular calcium, is responsible for the Ca 2ϩ independence of iNOS (5). Whether a given NOS isoform binds CaM in a Ca 2ϩ -dependent or -independent manner has been assumed to be a property solely of the amino acid sequence specified by a 20 -25-amino acid CaM binding site. However, this restrictive view is challenged by findings that ...

The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20 -40 residues longer in the C terminus, forming a "tail" that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7-21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.Nitric-oxide synthases (NOSs) 1 are bidomain, dimeric enzymes that synthesize NO from L-arginine through a series of monooxygenation reactions (for review see Ref. 1). The three isoforms, neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), produce NO by the same mechanism but play very different physiological roles due to the type of cell where they are located. nNOS, located in neurons in the brain and at neuromuscular junctions, is involved in neurotransmission (2, 3); iNOS, located in macrophages, is involved in the immune response (4, 5); and eNOS, located in endothelial cells, is involved in hemodynamic regulation (6, 7). The NO produced by nNOS and eNOS exerts its effects through the stimulation of guanylate cyclase, whereas the NO produced by iNOS exerts its effects directly or by combining with superoxide anion radical to form peroxynitrite anion, both potent oxidants deleterious to proteins and DNA.The NOSs consist of two domains, a heme and H 4 B-containing oxygenase (or heme) domain, which binds the substrate L-arginine, and a flavin-containing reductase (or flavoprotein) domain, which binds the prosthetic group flavins FAD and FMN and the cofactor NADPH. Electrons are transferred into NOS at the FAD moiety and are subsequently passed through the FMN to the heme domain. A calmodulin-binding region bisects the two domains. Calmodulin (CaM) is required for NO production and mediates the transfer of electrons from the FMN of NOS to the heme domain (8). CaM binds NOS in a 1:1 stoichiometry with very high affinity. CaM binds to sequences within the nNOS, eNOS, and iNOS CaM-binding...

In the absence of L-arginine, the heme center of the oxygenase domain of neuronal nitric-oxide synthase reduces molecular oxygen to superoxide (O 2 .

The nitric oxide synthases (NOS-I, neuronal, NOS-II, inducible, and NOS-III, endothelial) are the most recent additions to the large number of heme proteins that contain cysteine thiolate-liganded protoporphyrin IX heme prosthetic groups. This group of oxygenating enzymes also includes one of the largest gene families, that of the cytochromes P450, which have been demonstrated to be involved in the hydroxylation of a variety of substrates, including endogenous compounds (steroids, fatty acids, and prostaglandins) and exogenous compounds (therapeutic drugs, environmental toxicants, and carcinogens). The substrates for cytochromes P450 are universally hydrophobic while the physiological substrate for the nitric oxide synthases is the amino acid L-arginine, a hydrophilic compound. This review will discuss the approaches being used to study the structure and mechanism of neuronal nitric oxide synthase in the context of its known prosthetic groups and regulation by Ca(2+)-calmodulin and/or tetrahydrobiopterin (BH4).

W e describe an assay to measure the extent of enzymatic unwinding of D N A by a D N A helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the D N A unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono-and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. when corrected for observed stoichiometry]. With increasing NaCl concentration, k,, peaks a t 100 mM, and the apparent K , value for dsDNA increases by 3-fold a t 200 m M NaC1. In the presence of 5 m M calcium acetate, the apparent K , value is increased by 3-fold, and k,, decreased by 20-30%. W e have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule. R e c B C D enzyme is a 330-kDa protein consisting of three nonidentical subunits with five different activities. It has DNA-dependent ATPase, single-stranded DNA (ssDNA)' and double-stranded DNA (dsDNA) exonuclease, ssDNA endonuclease, and DNA helicase activities [for reviews, see Telander-Muskavitch and Linn (1981) and Taylor (1988)l. Escherichia coli mutants lacking the recBCD enzyme show a phenotypic reduction in recombination after conjugation or generalized transduction by as much as 3 orders of magnitude and exhibit a reduced ability to recover from DNA damage. Together, recBCD enzyme and recA protein define the RecBCD pathway of recombination, the major pathway used by E. coli. RecBCD enzyme has also been shown to nick dsDNA very specifically at x sites (Taylor et al., 1985), major recombinational uhotspots" [for a review of x sites, see Stahl The nuclease activities of recBCD enzyme are inhibited to varying degrees by SSB protein, which binds to ssDNA and protects it from recBCD enzyme nuclease (Mackay & Linn, 1976), by the presence of calcium ion (Rosamond et al., 1979), and by high levels of ATP. The degradation of dsDNA by recBCD enzyme requires ATP and exhibits an optimum at 20 pM ATP, where the final products of the reaction are single-stranded oligonucleotides three to eight bases long (in the absence of SSB protein and calcium ions) (Eichler & Lehman, 1977;Goldmark & Linn, 1972). The extent of exonuclease activity is inhibited at high (>200 pM) concentrations of ATP while the initial rate of nuclease activity remains unaffected. The products of the reaction under conditions of high ATP concentration are pieces of ssDNA with lengths ranging from 135 to 1400 nucleotides (Mackay & Linn, 1976). This difference in products at low and high ATP concentrations led Mackay and Linn (1976) to hypothesize ...