On the activation of soluble guanylyl cyclase by nitric oxide

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Nitric oxide (NO) exerts physiological effects by activating specialized receptors that are coupled to guanylyl cyclase activity, resulting in cGMP synthesis from GTP. Despite its widespread importance as a signal transduction pathway, the way it operates is still understood only in descriptive terms. The present work aimed to elucidate a formal mechanism for NO receptor activation and its modulation by GTP, ATP, and allosteric agents, such as YC-1 and BAY 41-2272. The model comprised a module in which NO, the nucleotides, and allosteric agents bind and the protein undergoes a conformational change, dovetailing with a catalytic module where GTP is converted to cGMP and pyrophosphate. Experiments on NO-activated guanylyl cyclase purified from bovine lung allowed values for all of the binding and isomerization constants to be derived. The catalytic module was a modified version of one describing the kinetics of adenylyl cyclase. The resulting enzyme-linked receptor mechanism faithfully reproduces all of the main functional properties of NO-activated guanylyl cyclase reported to date and provides a thermodynamically sound interpretation of those properties. With appropriate modification, it also replicates activation by carbon monoxide and the remarkable enhancement of that activity brought about by the allosteric agents. In addition, the mechanism enhances understanding of the behavior of the receptor in a cellular setting. Nitric oxide (NO)4 is normally generated mainly by endothelial and nerve cells and subserves an intercellular messenger role in most tissues, producing diverse effects, such as smooth muscle relaxation, inhibition of platelet aggregation, and synaptic plasticity (1, 2). Physiological NO signals are transduced through receptors equipped with guanylyl cyclase (GC) activity, leading to the intracellular formation of the effector molecule, cGMP (3). The discovery in homogenates of a "soluble" GC (4) that could be activated by NO (5) was vital for the identification of NO as an endogenous signaling agent. Nevertheless, despite many years of study, the mechanism of NO signal transduction through this pathway and the ways it is regulated remain indistinct. This is disappointing, because, as exemplified by synaptic transmission (6, 7), a quantitative understanding of how cells decode incoming signals provides powerful insight into the language of chemical communication and helps pinpoint abnormalities in disease states.In the current scheme (Fig. 1), the NO binding site is a heme prosthetic group, the occupation of which results in disengagement of a coordinating histidine bond, which helps drive a conformational change that propagates to the catalytic domain, resulting in the conversion of GTP into cGMP and pyrophosphate. Armed with spectroscopic data on the rates of binding and dissociation of [8][9][10] and with functional data on the potency and efficacy of NO and the rates at which cGMP synthesis activates and deactivates (11-18), it has been possible to insert values for the rates of the binding ...

Section: Methodsmentioning

confidence: 99%

An Enzyme-linked Receptor Mechanism for Nitric Oxide-activated Guanylyl Cyclase

Roy¹,

Halvey²,

Garthwaite

2008

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“…Recent structural studies suggested that NO binds to the proximal side of the heme in addition to the distal side thereby promoting desensitization (8 -11). However, the physiological significance of this alternate proximal binding of NO to the heme remains controversial (12)(13)(14)(15).…”

mentioning

confidence: 99%

Nitric Oxide-dependent Allosteric Inhibitory Role of a Second Nucleotide Binding Site in Soluble Guanylyl Cyclase

Chang

Lemme²,

Sun

et al. 2005

The mechanism of desensitization of the nitric oxide (NO) receptor (␣ 1 ⅐␤ 1 isoform of soluble guanylyl cyclase, sGC) is not known. Models of the structure of ␣ 1 ⅐␤ 1 , based on the x-ray crystal structure of adenylyl cyclase (AC) suggest the existence of a nucleotide-like binding site, in addition to the putative catalytic site. We have previously reported that mutating residues that coordinate Mg 2؉ GTP (substrate) binding in ␣ 1 ⅐␤ 1 into those present in AC fully reverts GC activity to AC activity. The wild-type form of ␣ 1 ⅐␤ 1 (GC-wt) and the mutant form (AC-mut, ␣ 1 R592Q⅐␤ 1 E473K,C541D) were purified, and their sensitivities to various nucleotides were assessed. In using the AC-mut as well as other mutants that coordinate purine binding, we were able to distinguish allosteric inhibitory effects of guanine nucleotides from competitively inhibitory effects on catalytic activity. Here we report that several nucleotide analogs drastically alter sGC and AC-mut activity by acting at a second nucleotide site, likely pseudosymmetric to the catalytic site. In particular, Mg 2؉ GTP␥S and Mg 2؉ ATP␥S inhibited cyclase activity through a mixed, non-competitive mechanism that was only observable under NO stimulation and not under basal conditions. The non-competitive pattern of inhibition was not present in mutants carrying the substitution ␤ 1 D477A, the pseudosymmetric equivalent to ␣ 1 D529 (located in the substrate-binding site and involved in substrate binding and catalysis), or with the double mutations ␣ 1 E525K,C594D, the pseudosymmetric equivalent to ␤ 1 E473K,C541D. Taken together these data suggest that occupation of the second site by nucleotides may underlie part of the mechanism of desensitization of sGC.

“…Despite the widely recognized importance of NO, little is known about the mechanism of regulation of the NO receptor, the soluble guanylyl cyclase (sGC) 1 (1)(2)(3). sGC is a heterodimeric enzyme formed by an ␣ and a ␤ subunit, the latter containing the heme where NO binds.…”

mentioning

confidence: 99%

Characterization of a Novel Type of Endogenous Activator of Soluble Guanylyl Cyclase

Balashova

Chang

Lamothe

et al. 2005

Nitric oxide (NO) remains the only firmly established endogenous modulator of soluble guanylyl cyclase (sGC) activity, but physiological, structural, and biochemical evidence now suggests that in vivo regulation of sGC involves direct interaction with other factors. We searched for such endogenous modulators in human umbilical vein endothelial cells and COS-7 cells. The cytosolic fraction of both cell types stimulated the activity of semipurified sGC severalfold in the absence or presence of a saturating concentration of NO. The cytosolic factor was sensitive to proteinase K and destroyed by boiling, suggesting that it contains a protein component. Size exclusion chromatography revealed peaks of activity between 40 and 70 kDa. The sGC-activating effect was further purified by ion exchange chromatography. In the presence of the benzylindazole YC-1 or NO, the partially purified factor synergistically activated sGC, suggesting that this factor had a mode of activation different from that of YC-1 or NO. Four candidate activators were identified from the final purification step by matrix-assisted laser desorption ionization mass spectrometry analysis. Using an sGC affinity matrix, one of them, the molecular chaperone Hsp70, was shown to directly interact with sGC. This interaction was further confirmed by co-immunoprecipitation in lung tissues and by co-localization in smooth muscle cells. sGC and Hsp70 co-localized at the plasma membrane, supporting the idea that sGC can be translocated to the membrane. Hsp70 co-purifies with the sGC-activating effect, and immunodepletion of Hsp70 from COS-7 cytosol coincided with a marked attenuation of the sGC-activating effect, yet the effect was not rescued by the addition of pure Hsp70. Thus, Hsp70 is a novel sGC-interacting protein that is responsible for the sGC-activating effect, probably in association with other factors or after covalent modification.