Michael A. Marletta

Nitric Oxide Synthase: Aspects Concerning Structure and Catalysis

Michael A. Marletta Interdepartmental Program in Medicinal Chemistry College of Pharmacy and Department of Biological Chemistry School of Medicine The University of Michigan Ann Arbor, Michigan 46109-l 065 The rapid development of our understanding of the biologi- cal actions of nitric oxide (NO) has, to a large degree, been paralleled by our understanding of the enzyme responsi- ble for the synthesis of NO, nitric oxide synthase (NOS). The relatively fast pace of advance in NOS enzymology is primarily due to the fact that structure/function questions have crossed over several well-established enzymatic problems. As will be outlined below, NOS is a complex enzyme involving several tightly bound redox cofactors that are apparently organized into discrete domains that can be associated with a particular activity. First, the en- zyme has significant homology to NADPH cytochrome P-450 reductase and has been shown to contain a cyto- chrome P-450-type heme and to carry out P-450 chemistry in the formation of NO. What then is the relationship of NOS to the large class of cytochrome P-450 isoenzymes? Second, constitutive NOS isoforms require Ca*+ and cal- modulin (CaM) while inducible NOS (INOS) shows no re- quirement, although these isoforms apparently have CaM as a tightly bound subunit. What is the nature of this im- portant difference in the recognition and binding of CaM? NOS also has a tightly associated reduced pterin that is very important for an enzyme activity whose function is still not known. Third, the product of the reaction, NO, typically is a strong heme ligand. How is it that the enzyme escapes self-inactivation during turnover? These ques- tions shall be the main the focus of this review. General Characteristics of the Reaction The reaction catalyzed by NOS is illustrated in Figure 1. As shown, the reaction requires molecular oxygen (02) and reducing equivalents in the form of NADPH (Marletta, 1993). Except for some minor structural modifications, all NOS isoforms specifically utilize L-arginine as the sub- strate. The products of the reaction are NO and citrulline (presumed to be the L-isomer). It is assumed, given the monooxygenase-like activity of NOS and the heavy iso- tope labeling studies, that H20 is the ultimate fate of the other oxygen atom. The initial NOS cDNA isolated was that of the neuronal isoform from rat cerebellum, where sequence analysis showed a significant homology of NADPH cytochrome P-450 reductase to the C-terminal sequence of this NOS isoform (Bredt et al., 1991). All clones isolated subsequently have demonstrated the same homology. Given that the normal function of this reductase is to supply reducing equivalents to cytochrome P-450, it has been assumed that this domain in NOS serves the same function. The findings that NOS also con-

Guanylate cyclase and the .NO/cGMP signaling pathway.

Signal transduction with the diatomic radical nitric oxide (NO) is involved in a number of important physiological processes, including smooth muscle relaxation and neurotransmission. Soluble guanylate cyclase (sGC), a heterodimeric enzyme that converts guanosine triphosphate to cyclic guanosine monophosphate, is a critical component of this signaling pathway. sGC is a hemoprotein; it is through the specific interaction of NO with the sGC heme that sGC is activated. Over the last decade, much has been learned about the unique heme environment of sGC and its interaction with ligands like NO and carbon monoxide. This review will focus on the role of sGC in signaling, its relationship to the other nucleotide cyclases, and on what is known about sGC genetics, heme environment and catalysis. The latest understanding in regard to sGC will be incorporated to build a model of sGC structure, activation, catalytic mechanism and deactivation.

Nitric oxide: biosynthesis and biological significance

The recent discovery that mammalian cells can synthesize nitric oxide coincided with the identification of this simple gas as a factor involved in cellular communication. Nitric oxide has now been shown to be derived from L-arginine in macrophages, endothelial cells and possibly other cell types. Its physiological role in macrophages may be as a cytotoxic agent. However, nitric oxide produced by endothelial cells is thought to trigger vascular smooth muscle relaxation through activation of the enzyme guanylate cyclase.

Catalysis by nitric oxide synthase

The enzyme nitric oxide synthase catalyzes the oxidation of the amino acid L-arginine to L-citrulline and nitric oxide in an NADPH-dependent reaction. Nitric oxide plays a critical role in signal transduction pathways in the cardiovascular and nervous systems and is a key component of the cytostatic/cytotoxic function of the immune system. Characterization of nitric oxide synthase substrates and cofactors has outlined the broad details of the overall reaction and suggested possibilities for chemical steps in the reaction; however, the molecular details of the reaction mechanism are still poorly understood. Recent evidence suggests a role for the reduced bound pterin in the first step of the reaction--the hydroxylation of L-arginine.

Nitric oxide: Cytokine-regulation of nitric oxide in host resistance to intracellular pathogens

To discover how nitric oxide (NO) synthesis is controlled in different tissues as cells within these tissues combat intracellular pathogens, we examined three distinctively different experimental murine models designed for studying parasite-host interactions: macrophage killing of Leishmania major; nonspecific protection against tularemia (Francisella tularensis) by Mycobacterium bovis (BCG); and specific vaccine-induced protection against hepatic malaria with Plasmodium berghei. Each model parasite and host system provides information on the source and role of NO during infection and the factors that induce or inhibit its production. The in vitro assay for macrophage antimicrobial activity against L. major identified cytokines involved in regulating NO-mediated killing of this intracellular protozoan. L. major induced the production of two competing cytokines in infected macrophages: (1) the parasite activated the gene for tumor necrosis factor (TNF), and production of TNF protein was enhanced by the presence of interferon-gamma (IFN-gamma). TNF then acted as a autocrine signal to amplify IFN-gamma-induced production of NO; and (2) the parasite upregulated production of transforming growth factor-beta (TGF-beta), which blocked IFN-gamma-induced production of NO. Whether parasite-induced TNF (parasite destruction) or TGF-beta (parasite survival) prevailed depended upon the presence and quantity of IFN-gamma at the time of infection. The relationship between NO production in vivo and host resistance to infection was demonstrated with M. bovis (BCG).(ABSTRACT TRUNCATED AT 250 WORDS)

Synergistic activation of soluble guanylate cyclase by YC-1 and carbon monoxide: implications for the role of cleavage of the iron-histidine bond during activation by nitric oxide

BACKGROUNDnNitric oxide (.NO) is used in biology as both an intercellular signaling agent and a cytotoxic agent. In signaling, submicromolar quantities of .NO stimulate the soluble isoform of guanylate cyclase (sGC) in the receptor cell. .NO increases the Vmax of this heterodimeric hemoprotein up to 400-fold by interacting with the heme moiety of sGC to form a 5-coordinate complex. Carbon monoxide (CO) binds to the heme to form a 6-coordinate complex, but only activates the enzyme 5-fold, YC-1 is a recently discovered compound that relaxes vascular smooth muscle by stimulating sGC.nnnRESULTSnIn the presence of YC-1, CO activates sGC to the same specific activity as attained with .NO. YC-1 did not affect the NO-stimulated activity. The on-rate (kon) and off-rate (koff) of CO for binding to sGC in the presence of YC-1 were determined by stopped-flow spectrophotometry. Neither the kon nor the koff varied from values previously obtained in the absence of YC-1, indicating that YC-1 has no effect on the affinity of CO for the heme. In the presence of YC-1, the visible spectrum of the sGC-CO complex has a Soret peak at 423 nm, indicating the complex is 6-coordinate.nnnCONCLUSIONSnYC-1 has no effect on the affinity of CO for the heme of sGC. In the presence of YC-1, maximal activation of sGC by CO is achieved by formation of a 6-coordinate complex between CO and the heme indicating that cleavage of the Fe-His bond is not required for maximal activation of sGC.

Mycobacteria Inhibit Nitric Oxide Synthase Recruitment to Phagosomes during Macrophage Infection

ABSTRACT Inducible nitric oxide synthase (iNOS) is a cytoplasmic protein responsible for the generation of nitric oxide (NO · ) in macrophages. In this work, we hypothesized that the intracellular localization of iNOS is significant for effective delivery of NOu2009·u2009 to phagosomes containing ingested microorganisms. Using immunofluorescence microscopy and Western blot analysis, iNOS was shown to localize in the vicinity of phagosomes containing latex beads in stimulated macrophages. iNOS also localized to phagosomes containing Escherichia coli. The colocalization of iNOS with ingested latex beads was an actin-dependent process, since treatment with the actin microfilament disrupter cytochalasin D prevented iNOS recruitment to latex bead phagosomes. In contrast to E. coli and inert particle phagosomes, mycobacterial phagosomes did not colocalize with iNOS. This study demonstrates that (i) iNOS can be recruited to phagosomes; (ii) this recruitment is dependent on a functional actin cytoskeleton; (iii) certain microorganisms have the ability to prevent or reduce colocalization with iNOS; and (iv) spatial exclusion of iNOS may play a role in Mycobacterium tuberculosis pathogenesis.

Inactivation of macrophage nitric oxide synthase activity by NG-Methyl-L-arginine

.N = O synthase catalyzes the oxidation of one of the two chemically equivalent guanido nitrogens of L-arginine to nitric oxide (.N = O). NG-Methyl-L-arginine has been previously characterized as a potent competitive inhibitor of both major types of .N = O synthases. Initial rate kinetics were performed with a spectrophotometric assay based on the oxidation of oxy- to methemoglobin by .N = O. NG-Methyl-L-arginine was a competitive inhibitor of .N = O synthase activity derived from activated murine macrophages with a Ki of 6.2 microM. When the enzyme was pre-incubated in the presence of the required cofactors NADPH and tetrahydrobiopterin, time- and concentration-dependent irreversible inactivation of the activity was observed. At 37 degrees C the kinact was 0.050 min-1. This inactivation process exhibited substrate protection, saturation kinetics and required the cofactors necessary for enzymatic turnover. These data indicate that NG-methyl-L-arginine acts as a mechanism-based enzyme inactivator of murine macrophage .N = O synthase.

The activation of neuronal NO synthase is mediated by G-protein βγ subunit and the tyrosine phosphatase SHP-2

In CHO cells we had found that CCK positively regulated cell proliferation via the activation of a soluble guanylate cyclase. Here we demonstrate that CCK stimulated a nitric oxide synthase (NOS) activity. The production of NO was involved in the proliferative response elicited by CCK regarding the inhibitory effect of NOS inhibitors L‐NAME and α‐guanidinoglutaric acid. We identified the NOS activated by the peptide as the neuronal isoform: the expression of the C415A neuronal NOS mutant inhibited both CCK‐induced stimulation of NOS activity and cell proliferation. These two effects were also inhibited after expression of the C459S tyrosine phosphatase SHP‐2 mutant and the βARKl (495–689) sequestrant peptide, indicating the requirement of activated SHP‐2 and G‐βγ subunit. Kinetic analysis (Western blot after coimmunoprecipitation and specific SHP‐2 activity) revealed that in response to CCK‐treatment, SHP‐2 associated to G‐β1 subunit, became activated, and then dephosphorylated the neuronal NOS through a direct association. These data demonstrate that the neuronal NOS is implicated in proliferative effect evoked by CCK. A novel growth signaling pathway is described, involving the activation of neuronal NOS by dephosphorylation of tyrosyl residues.—Cordelier, P., Estève, J.‐P., Rivard, N., Marletta, M., Vaysse, N., Susini, C., Buscail, L. The activation of neuronal no synthase is mediated by G‐protein βγ subunit and the tyrosine phosphatase SHP‐2. FASEB J. 13, 2037–2050 (1999)

Conformationally-restricted arginine analogues as alternative substrates and inhibitors of nitric oxide synthases

Conformationally restricted arginine analogues (1-5) were synthesized and found to be alternative substrates or inhibitors of the three isozymes of nitric oxide synthase (NOS). A comparison of k(cat)/Km values shows that (E)-3,4-didehydro-D,L-arginine (1) is a much better substrate than the corresponding (Z)-isomer (2) and 3-guanidino-D,L-phenylglycine (3), although none is as good a substrate as is arginine; 5-keto-D,L-arginine (4) is not a substrate, but is an inhibitor of the three isozymes. Therefore, it appears that arginine binds to all of the NOS isozymes in an extended (E-like) conformation. None of the compounds exhibits time-dependent inhibition of NOS, but they are competitive reversible inhibitors. Based on the earlier report that N(omega)-propyl-L-arginine is a highly selective nNOS inhibitor (Zhang, H. Q.; Fast, W.; Marletta, M.; Martasek, P.; Silverman, R. B. J. Med. Chem. 1997, 40, 3869), (E)-N(omega)-propyl-3,4-didehydro-D,L-arginine (5) was synthesized, but it was shown to be weakly potent and only a mildly selective inhibitor of NOS. Imposing conformational rigidity on an arginine backbone does not appear to be a favorable approach for selective NOS inhibition.