Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.2 (guanylate cyclase)
8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We present ab-initio density functional theory studies on the interactions of small biologically active molecules, namely NO, CO, O(2), H(2)O, and NO(2) (-) with the full-size heme group. Our results show that the small molecule-iron bond is the strongest in carbonyl and the weakest in nitrite system. Trans influence induced by NO binding to the five-coordinate heme complex is shown. Nitric oxide in the resulting complex might be described as NO(-). The differences among the small ligands of XO type (CO, NO, O(2)), and their distant chemical behavior from H(2)O and NO(2) (-) ligands in binding to the Fe(II) ion, are shown. Moreover, the role of the heme ring as a reservoir of electrons in the studied complexes is invoked. The analysis of the parameters defining the iron-histidine bond indicates that this bond is longer and weaker in nitrosyl and carbonyl complexes than in the other systems. Our findings support the proposed mechanism of soluble guanylate cyclase (sGC) activation and suggest that the first step of sGC activation by CO may be the same as during the activation by NO. Obtained results are then compared with the data concerning smaller model of the heme, the porphyrin complexes, available in the literature.
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PMID:Theoretical density functional theory studies on interactions of small biologically active molecules with isolated heme group. 1722 31

Determinants of the Fe-CO and C-O stretching frequencies in (imidazole)heme-CO adducts have been investigated via density functional theory (DFT) analysis, in connection with puzzling characteristics of the heme sensor protein CooA and of the H-NOX (Heme-Nitric Oxide and/or OXygen binding) family of proteins, including soluble guanylate cyclase (sGC). The computations show that two mechanisms of Fe-histidine bond weakening have opposite effects on the nuFeC/nuCO pattern. Mechanical tension is expected to raise nuFeC with little change in nuCO whereas the weakening of H-bond donation from the imidazole ligand has the opposite effect. Data on CooA indicate imidazole H-bond weakening associated with heme displacement, as part of the activation mechanism. The computations also reveal that protein-induced distortion of the porphyrin ring, a prominent structural feature of the H-NOX protein TtTar4H (Thermoanaerobacter tengcongensis Tar4 protein heme domain), has surprisingly little effect on nuFeC or nuCO. However, another structural feature, strong H-bonding to the propionates, is suggested to account for the weakened back bonding that is evident in sGC. TtTar4H-CO itself has an elevated nuFeC, which is successfully modeled as a compression effect, resulting from steric crowding in the distal pocket. nuFeC/nuCO data, in conjunction with modeling, can provide valuable insight into mechanisms for heme-protein modulation.
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PMID:DFT analysis of axial and equatorial effects on heme-CO vibrational modes: applications to CooA and H-NOX heme sensor proteins. 1821 76

Soluble guanylate cyclase (sGC) is an important heme sensor protein. Regulation of the status of heme in the heme binding domain (or HNOX domain) by various gaseous activators can increase the catalytic efficiency of the cyclase domain. Several studies have demonstrated that the full activation of sGC is directly related to the cleavage of the Fe-His bond of the HNOX domain. To expand the primary response of the sGC HNOX domain to the cleavage event, a structural model of the sGC HNOX domain was constructed using homology modeling and the Fe-His bond was released at 6 ns of a 10-ns molecular dynamics simulation. An instant increment of Calpha-RMSD over L2 (Loop2, residues 124-130) was found after the cleavage of the Fe-His bond, which was consistent with the principle component analysis (PCA). The energy analysis results suggest that the motions of L2 are energetic. Based on the results, energetic conformational transformation of L2 is identified as the primary response of the sGC HNOX domain to the cleavage of the Fe-His bond.
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PMID:Primary response of the sGC heme binding domain to the cleavage of the Fe-His bond. 1847 82

Soluble guanylyl/guanylate cyclase (sGC), the primary biological receptor for nitric oxide, is required for proper development and health in all animals. We have expressed heterodimeric full-length and N-terminal fragments of Manduca sexta sGC in Escherichia coli, the first time this has been accomplished for any sGC, and have performed the first functional analyses of an insect sGC. Manduca sGC behaves much like its mammalian counterparts, displaying a 170-fold stimulation by NO and sensitivity to compound YC-1. YC-1 reduces the NO and CO off-rates for the approximately 100-kDa N-terminal heterodimeric fragment and increases the CO affinity by approximately 50-fold to 1.7 microm. Binding of NO leads to a transient six-coordinate intermediate, followed by release of the proximal histidine to yield a five-coordinate nitrosyl complex (k(6-5) = 12.8 s(-1)). The conversion rate is insensitive to nucleotides, YC-1, and changes in NO concentration up to approximately 30 microm. NO release is biphasic in the absence of YC-1 (k(off1) = 0.10 s(-1) and k(off2) = 0.0015 s(-1)); binding of YC-1 eliminates the fast phase but has little effect on the slower phase. Our data are consistent with a model for allosteric activation in which sGC undergoes a simple switch between two conformations, with an open or a closed heme pocket, integrating the influence of numerous effectors to give the final catalytic rate. Importantly, YC-1 binding occurs in the N-terminal two-thirds of the protein. Homology modeling and mutagenesis experiments suggest the presence of an H-NOX domain in the alpha subunit with importance for heme binding.
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PMID:Allostery in recombinant soluble guanylyl cyclase from Manduca sexta. 1851 59

Echinoderm sperm use cyclic nucleotides (CNs) as essential second messengers to locate and swim towards the egg. Sea urchin sperm constitute a rich source of membrane-bound guanylyl cyclase (mGC), which was first cloned from sea urchin testis by the group of David Garbers. His group also identified speract, the first sperm-activating peptide (SAP) to be isolated from the egg investment (or egg jelly). This decapeptide stimulates sperm mGC causing a fast transient increase in cGMP that triggers an orchestrated set of physiological responses including: changes in: membrane potential, intracellular pH (pHi), intracellular Ca2+ concentration ([Ca2+]i) and cAMP levels. Evidence from several groups indicated that cGMP activation of a K+ selective channel was the first ion permeability change in the signaling cascade induced by SAPs, and recently the candidate gene was finally identified. Each of the 4 repeated, 6 trans-membrane segments of this channel contains a cyclic nucleotide binding domain. Together they comprise a single polypeptide chain like voltage-gated Na+ or Ca2+ channels. This new type of channel, named tetraKCNG, appears to belong to the exclusive club of novel protein families expressed only in sperm and its progenitors. SAPs also induce fluctuations in flagellar [Ca2+]i that correlate with changes in flagellar form and regulate sperm trajectory. The motility changes depend on [Ca2+]i influx through specific Ca2+ channels and not on the overall [Ca2+]i in the sperm flagellum. All cilia and flagella have a conserved axonemal structure and thus understanding how Ca2+ regulates cilia and flagella beating is a fundamental question.
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PMID:Sperm-activating peptides in the regulation of ion fluxes, signal transduction and motility. 1864 73

Since the discovery of soluble guanylate cyclase (sGC) as the mammalian receptor for nitric oxide (NO), numerous studies have been performed in order to understand how sGC transduces the NO signal. However, the structural basis of sGC activation is still not completely elucidated. Spectroscopic and kinetic studies showed that the key step in the activation mechanism was the NO-induced breaking of the iron proximal histidine bond in the so-called 6c-NO to 5c-NO transition. The main breakthrough in the understanding of sGC activation mechanism came, however, from the elucidation of crystal structures for two different prokaryotic heme NO oxygen (HNOX) domains, which are homologues to the sGC heme domain. In this work we present computer simulation results of Thermoanaerobacter tencogensis HNOX that complement these structural studies, yielding molecular explanations to several poorly understood properties of these proteins. Specifically, our results explain the differential ligand binding patterns of the HNOX domains according to the nature of proximal and distal residues. We also show that the natural dynamics of these proteins is intimately related with the proposed conformational dependent activation process, which involves mainly the alphaFbeta1 loop and the alphaA-alphaC distal subdomain. The results from the sGC models also support this view and suggest a key role for the alphaFbeta1 loop in the iron proximal histidine bond breaking process and, therefore, in the sGC activation mechanism.
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PMID:Dynamical characterization of the heme NO oxygen binding (HNOX) domain. Insight into soluble guanylate cyclase allosteric transition. 1870 31

The iron complex of oxypyriporphyrin, a porphyrinoid containing a keto-substituted pyridine, was coupled with apomyoglobin. The reconstituted ferric myoglobin was found to be five-coordinate without iron-bound water molecules. The anionic ligands such as CN (-) and N 3 (-) bound the myoglobin with high affinities, while neutral imidazole did not. The IR observation indicated that the azide complex was pure high-spin, although the corresponding native protein was in the spin-state equilibrium. The reduced myoglobin was five-coordinate but exhibited no measurable affinity for O 2. The affinity for CO was lowered down to 1/2400 as compared with native myoglobin. These anomalies were ascribed to the deformation in the iron coordination core after the replacement of one of the four pyrroles with a larger pyridine ring. The ligand binding analyses for the ferric and ferrous myoglobin suggest that the proximal histidine pulls the iron atom from the deformed core to reduce the interaction between the iron and exogenous ligands. Similarity of the reconstituted myoglobin with guanylate cyclase, a NO-responsive signaling hemoprotein, was pointed out.
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PMID:Functional evaluation of iron oxypyriporphyrin in protein heme pocket. 1884 46

Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni(2+)-NTA, and POROS Q columns obtained approximately 100mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M(r)=144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS-PAGE corresponding to the alpha (M(r) approximately 77,000) and beta (M(r) approximately 70,000) sGC subunits. It showed an A(430)/A(280)=1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k(cat)/K(m)=1.7 x 10(-3)s(-1)microM(-1) that increased to 5.8 x 10(-1)s(-1)microM(-1) upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure-function relationships.
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PMID:Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells. 1918 60

Carbon monoxide (CO) is currently being evaluated as a therapeutic modality in the treatment of patients with acute lung injury and acute respiratory distress syndrome. No study has assessed the effects of CO on transepithelial ion transport and alveolar fluid reabsorption, two key aspects of alveolocapillary barrier function that are perturbed in acute lung injury/acute respiratory distress syndrome. Both CO gas (250 ppm) and CO donated by the CO donor, CO-releasing molecule (CORM)-3 (100 microM in epithelial lining fluid), applied to healthy, isolated, ventilated, and perfused rabbit lungs, significantly blocked (22)Na(+) clearance from the alveolar compartment, and blocked alveolar fluid reabsorption after fluid challenge. Apical application of two CO donors, CORM-3 or CORM-A1 (100 microM), irreversibly inhibited amiloride-sensitive short-circuit currents in H441 human bronchiolar epithelial cells and primary rat alveolar type II cells by up to 40%. Using a nystatin permabilization approach, the CO effect was localized to amiloride-sensitive channels on the apical surface. This effect was abolished by hemoglobin, a scavenger of CO, and was not observed when inactive forms of CO donors were employed. The effects of CO were not blocked by 8-bromoguanosine-3',5'-cyclic guanosine monophosphate, soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), or inhibitors of trafficking events (phalloidin oleate, MG-132, and brefeldin A), but the amiloride affinity of H441 cells was reduced after CO exposure. These data indicate that CO rapidly inhibits sodium absorption across the airway epithelium by cyclic guanosine monophosphate- and trafficking-independent mechanisms, which may rely on critical histidine residues in amiloride-sensitive channels or associated regulatory proteins on the apical surface of lung epithelial cells.
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PMID:Carbon monoxide rapidly impairs alveolar fluid clearance by inhibiting epithelial sodium channels. 1925 42

The bacterial heme protein Alcaligenes xylosoxidans cytochrome c' (AXCP) forms a novel five-coordinate heme-nitrosyl (5c-NO) complex in which NO resides at the proximal heme face in place of the endogenous protein ligand. Intriguingly, AXCP shares NO-binding properties with the eukaryotic NO-sensor, soluble guanylate cyclase (sGC), including 5c-NO formation via two NO-dependent reactions. For both proteins, a model has been proposed in which NO binds to the vacant distal face to form a transient six-coordinate heme-nitrosyl (6c-NO) species, which then converts to a proximal 5c-NO complex via a putative dinitrosyl intermediate. To shed light on this novel reaction mechanism, activation parameters have been determined for distal and proximal NO-binding reactions in AXCP from the effect of temperature and hydrostatic pressure on rate constants. The unusually slow 6c-NO formation reaction has a near-zero entropy of activation and a positive volume of activation (DeltaV(double dagger) = +14.1 cm(3) mol(-1)), consistent with a rate-determining step involving movement of the Leu 16 residue to allow NO binding to the crowded distal site. For the 6c-NO --> 5c-NO conversion, the large positive entropy of activation (DeltaS(double dagger) = +103 J K(-1) mol(-1)) and volume of activation (DeltaV(double dagger) = +24.1 cm(3) mol(-1)) suggest that the putative dinitrosyl intermediate forms via a dissociative mechanism in which the endogenous His ligand dissociates prior to the attack of the second NO molecule on the proximal heme face. These results have important implications for distal vs proximal NO binding in AXCP, as well as mechanisms of 5c-NO formation in heme proteins.
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PMID:Activation parameters for heme-NO binding in alcaligenes xylosoxidans cytochrome c': the putative dinitrosyl intermediate forms via a dissociative mechanism. 1933 78


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