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)

Soluble guanylate cyclase isolated from bovine and rat lung is a heterodimeric hemoprotein composed of alpha1 and beta1 subunits. The heme binding region has been localized to residues 1-385 of the beta1 subunit [beta1(1-385)], while the catalytic site(s) have been localized to the C-terminal region of sGC. There are four conserved histidine residues in the heme binding region of sGC. H220 and H346 are conserved among all known sGC subunits (alpha and beta), while H105 and H134 are conserved only in the beta subunits (beta1 and beta2). Site-directed mutagenesis was used to individually change each of the conserved histidines in sGC beta1(1-385) to alanine or glycine, and the resulting mutants were expressed in E. coli. All of the mutants except for H105A and H105G had heme bound as isolated. Imidazole (Im) was able to rescue heme binding to H105G when added to the growth medium and purification buffers. The heme in H105G isolated in the presence of imidazole [H105G(Im)] was ferric and a mixture of 5-coordinate, high-spin and 6-coordinate, low-spin complexes. After reduction, the ferrous heme in H105G(Im) was 5-coordinate, high-spin as indicated by resonance Raman spectroscopy. When imidazole in H105G(Im) was exchanged with N-methylimidazole (MeIm), the Fe-N(Im/MeIm) stretching frequency was shifted from 221 to 212 cm-1. A shift of this magnitude is expected when the ligand is directly coordinated to the heme iron. All of the data are consistent with the conclusion that H105 in the beta1 subunit is the heme proximal ligand.
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PMID:Identification of histidine 105 in the beta1 subunit of soluble guanylate cyclase as the heme proximal ligand. 952 70

When expressed in Escherichia coli, the heme domain [beta1(1-385)] of rat lung soluble guanylate cyclase (sGC) is isolated with a stoichiometric amount of bound heme [Zhao, Y., and Marletta, M. A. (1997) Biochemistry 36, 15959-15964]. Nitric oxide (NO) binding to the heme in beta1(1-385) leads to cleavage of the Fe-His bond and formation of a five-coordinate NO-heme complex. Addition of imidazole to the five-coordinate NO complex shifts the Soret peak from 399 to 420 nm, which appears to result from the formation of a six-coordinate NO complex. Removal of the added imidazole by gel filtration results in formation of the five-coordinate NO complex once again. The EPR spectrum of the putative six-coordinate NO complex has nine distinct derivative-shaped lines (a triplet of triplets), which is the signature spectrum of a six-coordinate NO complex with two nitrogen atoms as the axial ligands. [15N]Imidazole simplifies the six-coordinate NO complex EPR spectrum to six distinct derivative-shaped lines (a triplet of doublets), indicating that the other axial ligand in the six-coordinate NO complex is an imidazole molecule. These results show that NO binding to sGC not only leads to the cleavage of the Fe-His bond but also induces a conformational change which opens the heme proximal pocket large enough to accommodate an exogenous imidazole molecule. These observations have important implications for determining the NO activation mechanism of sGC.
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PMID:Structural changes in the heme proximal pocket induced by nitric oxide binding to soluble guanylate cyclase. 973 Aug 18

The nature of the metal-proximal base bond of soluble guanylate cyclase from bovine lung was examined by EPR spectroscopy. When the ferrous enzyme was mixed with NO, a new species was transiently produced and rapidly converted to a five-coordinate ferrous NO complex. The new species exhibited the EPR signal of six-coordinate ferrous NO complex with a feature of histidine-ligated heme. The histidine ligation was further examined by using the cobalt protoporphyrin IX-substituted enzyme. The Co2+-substituted enzyme exhibited EPR signals of a broad g perpendicular;1 component and a g;1 component with a poorly resolved triplet of 14N superhyperfine splittings, which was indicative of the histidine ligation. These EPR features were analogous to those of alpha-subunits of Co2+-hemoglobin in tense state, showing a tension on the iron-histidine bond of the enzyme. The binding of NO to the Co2+-enzyme markedly stimulated the cGMP production by forming the five-coordinate NO complex. We found that N3- elicited the activation of the ferric enzyme by yielding five-coordinate high spin N3- heme. These results indicated that the activation of the enzymes was initiated by NO binding to the metals and proceeded via breaking of the metal-histidine bonds, and suggested that the iron-histidine bond in the ferric enzyme heme was broken by N3- binding.
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PMID:EPR characterization of axial bond in metal center of native and cobalt-substituted guanylate cyclase. 1007 61

Previous work has proved that the enzyme-soluble guanylate cyclase, GC, is activated several 100-fold by the combination of carbon monoxide plus a benzylindazole derivative called YC-1. That is about the same as activation by nitric oxide, which has a well-established role both in vivo and in vitro. This report addresses several spectroscopic, equilibrium, and kinetic effects wrought by YC-1 on carboxyl guanylate cyclase, including the following: a shift in the Soret absorption band by 4 nm to shorter wavelength; an increase in CO affinity by an order of magnitude; a dramatic change in the kinetics of CO association. After photolytic dissociation of CO, the majority, but not all, of bimolecular ligand recombination occurs with a time constant about 1000-fold faster than in the absence of YC-1, while a smaller fraction recombines almost, but not quite, the same as usual. This is reminiscent of the kinetics of NO association with GC, which also shows two prominent phases. The results just listed pertain in the presence of GTP/cGMP, which would be present during enzyme catalysis. Qualitatively similar, but smaller, effects occur in the absence of GTP/cGMP. Measurements are reported to characterize other changes in buffer conditions. The results are consistent with a mechanistic model that attributes a crucial role to the proximal bond that connects the heme iron to a histidine side chain in GC but also requires protein control of the distal environment.
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PMID:Kinetics and equilibria of soluble guanylate cyclase ligation by CO: effect of YC-1. 1045 64

Soluble guanylyl cyclase acts as the receptor for the signaling molecule nitric oxide. The enzyme consists of two different subunits. Each subunit shows the cyclase catalytic domain, which is also conserved in the membrane-bound guanylyl cyclases and the adenylyl cyclases. The N-terminal regions of the subunits are responsible for binding of the prosthetic heme group of the enzyme, which is required for the stimulatory effect of nitric oxide (NO). The five-coordinated ferrous heme displays a histidine as the axial ligand; activation of soluble guanylyl cyclase by NO is initiated by binding of NO to the heme iron and proceeds via breaking of the histidine-to-iron bond. Recently, a novel pharmacological and possibly physiological principle of guanylyl cyclase sensitization was demonstrated. The substance YC-1 has been shown to activate the enzyme independent of NO, to potentiate the effect of submaximally effective NO concentrations, and to turn carbon monoxide into an effective activator of soluble guanylyl cyclase.
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PMID:Studying the structure and regulation of soluble guanylyl cyclase. 1058 Nov 48

Soluble guanylate cyclase (GC) from bovine lung is activated 4-fold by carbon monoxide (CO) and 400-fold by nitric oxide (NO). Spectroscopic and kinetic data for ligation of CO and NO with GC are summarized and compared with similar data for myoglobin (Mb), hemoglobin (Hb), and heme model compounds. Kinetic, thermodynamic, and structural data form a basis on which to construct a model for the manner in which the two ligands affect protein structure near the heme for heme proteins in general and for GC in particular. The most significant datum is that although association rates of ligands with GC are similar to those with Mb and Hb, their dissociation rates are dramatically faster. This suggests a delicate balance between five- and six-coordinate heme iron in both NO and CO complexes. Based on these and other data, a model for GC activation is proposed: The first step is formation of a six-coordinate species concomitant with tertiary and quaternary structural changes in protein structure and about a 4-fold increase in enzyme activity. In the second step, applicable to NO, the bond from iron to the proximal histidine ruptures, leading to additional relaxation in the quaternary and tertiary structure and a further 100-fold increase in activity. This is the main event in activation, available to NO and possibly other activators or combinations of activators. It is proposed, finally, that the proximal base freed in step 2, or some other protein base suitably positioned as a result of structural changes following ligation, may provide a center for nucleophilic substitution catalyzing the reaction GTP --> cGMP. An example is provided for a similar reaction in a derivatized protoheme model compound. The reaction mechanism attempts to rationalize the relative enzymatic activities of GC, heme-deficient GC, GC-CO, and GC-NO on a common basis and makes predictions for new activators that may be discovered in the future.
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PMID:Activation of soluble guanylate cyclase by carbon monoxide and nitric oxide: a mechanistic model. 1058 Nov 49

Nitric oxide (NO) functions as a signaling agent by activation of the soluble isoform of guanylate cyclase (sGC), a heterodimeric hemoprotein. NO binds to the heme of sGC and triggers formation of cGMP from GTP. Here we report direct kinetic measurements of the multistep binding of NO to sGC and correlate these presteady state events with activation of enzyme catalysis. NO binds to sGC to form a six-coordinate, nonactivated, intermediate (k(on) > 1.4 x 10(8) M(-1).s(-1) at 4 degrees C). Subsequent release of the axial histidine heme ligand is shown to be the molecular step responsible for activation of the enzyme. The rate at which this step proceeds also depends on NO concentration (k = 2.4 x 10(5) M(-1).s(-1) at 4 degrees C), thus identifying a novel mode of regulation by NO. NO binding to the isolated heme domain of sGC was also rapid (k = 7.1 +/- 2 x 10(8) M(-1).s(-1) at 4 degrees C); however, no intermediate was observed. The data show that sGC acts as an extremely fast, specific, and highly efficient trap for NO and that cleavage of the iron-histidine bond provides the driving force for activation of sGC. In addition, the kinetic data indicate that transport or stabilization of NO is not necessary for effective signal transmission.
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PMID:A molecular basis for nitric oxide sensing by soluble guanylate cyclase. 1061 Dec 85

Soluble guanylyl cyclase (sGC) is known to be activated by NO binding to the heme moiety; previous studies have shown that CO does not activate sGC to the same extent as NO. Resonance Raman spectroscopy reveals different heme pocket structures for soluble guanylyl cyclase prepared by alternate methods, all of which display activation by NO. In our preparation, and in the expressed protein sGC1, the resting Fe(II) state is mainly 6-coordinate and low-spin, and the CO adduct has vibrational frequencies characteristic of a histidine-heme-CO complex in a hydrophobic environment. In contrast, the protein sGC2 is 5-coordinate, high-spin in the resting state, and the CO adduct has perturbed vibrational frequencies indicative of a negatively polarizing residue in the binding pocket. The differences may result from the need to reconstitute sGC1 or different isolation procedures for sGC1 versus sGC2. However, both sGC1 and sGC2 are activated by the same mechanism, namely displacement of the proximal histidine ligand upon NO binding, and neither one is activated by CO. If CO is an activator in vivo, some additional molecular component is required.
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PMID:Variable forms of soluble guanylyl cyclase: protein-ligand interactions and the issue of activation by carbon monoxide. 1063 13

The objective of this study was to test the hypothesis that the dietary dipeptide carnosine (beta-alanine-L-histidine) causes direct decreases in arterial tone. Isolated descending thoracic aortic rings from male Sprague-Dawley rats were used for all studies. Preconstriction of vessels was accomplished with phenylephrine. Carnosine (0.625-20 mM) produced dose-dependent vascular relaxation (P < 0.05) that was independent of endothelium. The constituent amino acid L-histidine did not produce any significant relaxation over the same dose range, whereas beta-alanine actually produced dose-dependent vasoconstriction (P < 0.05). The soluble guanylate cyclase inhibitor methylene blue (10(-5) M) significantly decreased the relaxation produced by carnosine (P < 0.05). Measurement of cyclic GMP in the presence and absence of methylene blue after carnosine and phenylephrine exposure was also done. Methylene blue 10(-5) M resulted in a decrease in cyclic GMP levels from 65.3 +/- 15.6 fmol/mg protein to 8.6 +/- 0.9 fmol/mg of protein (P = 0.001). We conclude that carnosine produces relaxation of isolated rat aorta independent of endothelium. The effect of carnosine is at least in part mediated via cyclic GMP production and is not reproduced by its constituent amino acids, L-histidine and beta-alanine.
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PMID:Vasodilatory actions of the dietary peptide carnosine. 1070 70

The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO-independent fashion. In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone. We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 microM YC-1, the V(max)/K(m GTP) increases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO-bound form from 474 to 492 cm(-1). Similarly, YC-1 has no effect on the RR spectrum of ferrous beta1(1-385), the isolated sGC heme-binding domain, but shifts the nu(Fe-CO) of CO-beta1(1-385) from 478 to 491 cm(-1), indicating that YC-1 binds in heme-binding region of sGC. In addition, the CO-bound forms of sGC and beta1(1-385) in the presence of YC-1 lie on the nu(Fe-CO) vs nu(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does not shift nu(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of beta1(1-385). The data are consistent with binding of CO and YC-1 to the sGC heme-binding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket.
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PMID:Interaction of soluble guanylate cyclase with YC-1: kinetic and resonance Raman studies. 1074 11


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