Expression and Characterization of the Catalytic Domains of Soluble Guanylate Cyclase: Interaction with the Heme Domain
Abstract
The catalytic domains (alpha(cat) and beta(cat)) of alpha1beta1 soluble guanylate cyclase (sGC) were expressed in Escherichia coli and purified to homogeneity. alpha(cat), beta(cat), and the alpha(cat)beta(cat) heterodimeric complex were characterized by analytical gel filtration and circular dichroism spectroscopy, and activity was assessed in the absence and presence of two different N-terminal regulatory heme-binding domain constructs. Alpha(cat) and beta(cat) were inactive separately, but together the domains exhibited guanylate cyclase activity. Analysis by gel filtration chromatography demonstrated that each of the approximately 25-kDa domains form homodimers. Heterodimers were formed when alpha(cat) and beta(cat) were combined. Results from circular dichroism spectroscopy indicated that no major structural changes occur upon heterodimer formation. Like the full-length enzyme, the alpha(cat)beta(cat) complex was more active in the presence of Mn(2+) as compared to the physiological cofactor Mg(2+), although the magnitude of the difference was much larger for the catalytic domains than for the full-length enzyme. The K(M) for Mn(2+)-GTP was measured to be 85 +/- 18 microM, and in the presence of Mn(2+)-GTP, the K(D) for the alpha(cat)beta(cat) complex was 450 +/- 70 nM. The N-terminal heme-bound regulatory domain of the beta1 subunit of sGC inhibited the activity of the alpha(cat)beta(cat) complex in trans, suggesting a domain-scale mechanism of regulation by NO. A model in which binding of NO to sGC causes relief of an autoinhibitory interaction between the regulatory heme-binding domain and the catalytic domains of sGC is proposed.
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Other studies show impaired recovery in peripheral artery disease patients with l-arginine.
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From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [1014]
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- Authors
- Winger, J. A., & Marletta, M. A.
- Affiliations
- Lawrence Berkeley National Laboratory (US)
- Corresponding author
- Marletta, Michael A.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2005-02-17
- Volume
- 44
- Issue
- 10
- Pages
- 4083-4090
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2024198419
- Cited by
- 87
- References
- 38
- Citations by year
- 2025: 1 · 2023: 2 · 2021: 2 · 2020: 3 · 2019: 4 · 2018: 6 · 2017: 6 · 2016: 4 · 2015: 6 · 2014: 7 · 2013: 17 · 2012: 3
- FWCI
- 1.63
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (T32GM007767, GM-07767)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Hemoglobin structure and function
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
- Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
- Keywords
- soluble guanylate cyclase, catalytic domain, heme-binding domain, heterodimer formation, homodimerization, manganese cofactor, magnesium cofactor, GTP binding, nitric oxide regulation, autoinhibition, enzyme inhibition
- MeSH terms
- Animals, Enzyme Activation, Escherichia coli, Guanosine Triphosphate, Guanylate Cyclase, Heme, Peptide Fragments, Protein Binding, Solubility, Substrate Specificity, Protein Structure, Tertiary, Dimerization, Catalytic Domain, Protein Subunits, Rats
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