A constitutively activated mutant of human soluble guanylyl cyclase (sGC): Implication for the mechanism of sGC activation

Martin, Emil; Sharina, Iraida; Kots, Alexander Y.; Murad, Ferid

doi:10.1073/pnas.1633590100

Cited by 62 publications

(62 citation statements)

References 40 publications

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“…PPIX effectively activates the enzyme in the absence of any gaseous ligands by replacing the ferrous heme moiety (19). Low concentrations of Tween 20 facilitates this replacement (17,20) by promoting heme depletion. As demonstrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Assay of sGC Activity-Enzyme activity was assayed by formation of [ 32 P]cGMP from [␣-32 P]GTP at 37°C as described previously (17). Briefly, the reaction was initiated by addition of 1 mM GTP to the enzyme in 25 mM TEA (pH 7.5), 1 mg/ml bovine serum albumin, 1 mM 3-isobutyl-1-methylxanthine, 1 mM cGMP, 3 mM MgCl 2 , 0.05 mg/ml creatine phosphokinase, and 5 mM creatine phosphate.…”

Section: Methodsmentioning

confidence: 99%

“…Dilutions of 2-(N,N-diethylamino)diazenolate-2-oxide diethylammonium salt (DEA-NO) donor were prepared in 10 mM NaOH and added to the enzyme immediately before starting the reaction with GTP. The enzyme was incubated with GTP at 37°C for 4 min, stopped by zinc acetate, and processed as described previously (17). To measure the extent of protoporphyrin IX (PPIX) activation, the Sf9 lysates containing wild-type or mutant enzyme were treated with 0.2% Tween 20 for 10 min on ice and then added to the reaction buffer containing various concentrations of PPIX.…”

Section: Methodsmentioning

confidence: 99%

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Ligand Selectivity of Soluble Guanylyl Cyclase

Martin

Berka²,

Bogatenkova

et al. 2006

Journal of Biological Chemistry

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Although soluble guanylyl cyclase (sGC) functions in an environment in which O 2 , NO, and CO are potential ligands for its heme moiety, the enzyme displays a high affinity for only its physiological ligand, NO, but has a limited affinity for CO and no affinity for O 2 . Recent studies of a truncated version of the sGC ␤ 1 -subunit containing the heme-binding domain (Boon, E. M., Huang, S H., and Marletta, M. A. (2005) Nat. Chem. Biol., 1, 53-59) showed that introduction of the hydrogen-bonding tyrosine into the distal heme pocket changes the ligand specificity of the heme moiety and results in an oxygen-binding sGC. The hypothesis that the absence of hydrogen-bonding residues in the distal heme pocket is sufficient to provide oxygen discrimination by sGC was put forward. We tested this hypothesis in a context of a complete sGC heterodimer containing both the intact ␣ 1 -and ␤ 1 -subunits. We found that the I145Y substitution in the full-length ␤-subunit of the sGC heterodimer did not produce an oxygen-binding enzyme. However, this substitution impeded the association of NO and destabilized the NO⅐heme complex. The tyrosine in the distal heme pocket also impeded both the binding and dissociation of the CO ligand. We propose that the mechanism of oxygen exclusion by sGC not only involves the lack of hydrogen bonding in the distal heme pocket, but also depends on structural elements from other domains of sGC. Soluble guanylyl cyclase (sGC)4 is a member of the guanylyl cyclase family of proteins, which respond to various ligands by converting GTP into cGMP. sGC stands apart from other members of its family by the nature of its activating ligand. Although other guanylyl cyclases are stimulated in response to various hormonal peptide ligands such as natriuretic peptides, guanylins, or toxins like heat-stable enterotoxin (see Ref. 1 for review), sGC is activated several hundredfold upon exposure to NO produced by nitric-oxide synthase. sGC is the key component of the NO/cGMP pathway and is crucial in mediating various physiological effects of NO such as blood vessel relaxation, inhibition of platelet aggregation, neurotransmission, and many others (2).The affinity of the sGC enzyme for NO is mediated by the ferrous heme prosthetic group. sGC possesses a ligand selectivity unique for hemeproteins. NO binds to the sGC heme at rates that are almost diffusion-limited (3) to form a stable NO⅐heme complex. High affinity coupled with high specificity for NO is essential for sGC to function as an NO receptor. Although sGC functions in the intracellular environment containing 20 -40 M O 2 and up to 10 nM NO during NO synthesis (4), sGC is capable of discriminating between these two similar molecules in favor of NO. Moreover, even in the absence of NO, sGC does not form an oxyferrous heme complex and does not show any significant autoxidation. This is especially interesting considering that, in some aspects, the sGC heme is similar to the heme moiety of globin oxygen carriers such as myoglobins and hemoglobins. Similar to globi...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Ligand Selectivity of Soluble Guanylyl Cyclase

Martin

Berka²,

Bogatenkova

et al. 2006

Journal of Biological Chemistry

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“…Activity of guanylyl cyclase was determined by RIA (22). Activity of purified human recombinant soluble guanylyl cyclase (23) (from E. Martin, University of Texas, Institute of Molecular Medicine, Houston, TX) was measured as described in ref. 24.…”

Section: Methodsmentioning

confidence: 99%

Pyridopyrimidine derivatives as inhibitors of cyclic nucleotide synthesis: Application for treatment of diarrhea

Kots¹,

Choi²,

Estrella-Jimenez

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…Some previous studies support the notion that an alternative coordination of sGC heme may be conceivable. For example, we have previously reported that, upon heme reconstitution, the mutant ␣␤Cys-105 sGC displayed spectral properties characteristic of a histidine-ligated hemoprotein, although the heme-coordinating His-105 was mutated to cysteine (42). These data suggest that the heme proximal ligand in the ␣␤H105C mutant is not Cys-105 but rather an alternative histidine, perhaps His-107.…”

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confidence: 97%

Dynamic Ligand Exchange in Soluble Guanylyl Cyclase (sGC)

Tsai

Berka

Sharina

et al. 2011

Journal of Biological Chemistry

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Background:The enzyme soluble guanylyl cyclase (sGC) converts the NO signal into cGMP. Results: Stoichiometric NO forms an unstable sGC-NO complex, which is stabilized by extra NO, GTP, or substitution of ␤His-107. Conclusion: Exchange of the proximal heme ligand contributes to sGC activation and desensitization. Significance: Understanding the dynamics of sGC/NO interaction and its functional consequences is necessary to understand the process of NO/cGMP signaling.

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A constitutively activated mutant of human soluble guanylyl cyclase (sGC): Implication for the mechanism of sGC activation

Cited by 62 publications

References 40 publications

Ligand Selectivity of Soluble Guanylyl Cyclase

Ligand Selectivity of Soluble Guanylyl Cyclase

Pyridopyrimidine derivatives as inhibitors of cyclic nucleotide synthesis: Application for treatment of diarrhea

Dynamic Ligand Exchange in Soluble Guanylyl Cyclase (sGC)

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