Functional characterization of two nucleotide-binding sites in soluble guanylate cyclase.

Full Abstract

Soluble guanylate cyclase is a heterodimeric hemoprotein composed of alpha- and beta-subunits with a homologous motif to the nucleotide-binding sites of adenylate cyclases. Homology modeling of guanylate cyclase, based on the crystal structure of adenylate cyclase, reveals a single GTP-binding site and a putative second site pseudosymmetric to the GTP-binding site. However, the role of this pseudosymmetric site has remained unclear. Using equilibrium dialysis, we identified two nucleotide-binding sites with high and low affinity for alpha,beta-methylene guanosine 5'-triphosphate (GMP-CPP). In contrast, 2'-dADP occupied both sites with equivalent affinities. Adenosine-5'-beta,gamma-imido triphosphate (AMP-PNP), which competitively inhibited the cyclase reaction, bound solely to the high affinity site, indicating the role of this site as the catalytic site. The function of the low affinity site was examined using allosteric activators YC-1 and BAY 41-2272. YC-1 significantly reduced the affinity of 2'-dADP, probably by competing for the same site as 2'-dADP. BAY 41-2272 totally inhibited the specific binding of one molecule of 2'-dADP as well as GMP-CPP. This suggests that the activators compete with these nucleotides for the low affinity site. Infrared and EPR analyses of the enzymic CO- and NO-hemes also supported the suggested role of the low affinity site as a target for the activators. Our results imply that the low affinity site is the pseudosymmetric site, which binds YC-1 or BAY 41-2272.

Learn Faster Today Improve your study skills

Author information

Author/s: Yazawa, Shinsuke (S); Tsuchiya, Hidemi (H); Hori, Hiroshi (H); Makino, Ryu (R);

Affiliation: Department of Life Science, College of Science, Rikkyo University, Nishi-ikebukuro 3-34-1, Toshima-ku, Tokyo 171-8501, Japan.

Journal and publication information

Publication Type: Journal Article; Research Support, Non-U.S. Gov't

Journal: The Journal of biological chemistry (J Biol Chem), published in United States. (Language: eng)

Reference: 2006-Aug; vol 281 (issue 31) : pp 21763-70

Dates: Created 2006/07/31; Completed 2006/10/16; Revised 2006/11/15;

PMID: 16754683, status: MEDLINE (last retrieval date: 12/26/2008)

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

External Links for this article (including full text providers, if available):

Click Electronic Full-text Provider Links to see options for finding the electronic full text links to this article. Note there may be a subscription or fee required for access to the full text. See our FAQ for information on finding FREE full text articles.

This article may also be located in paper journal collections available in many libraries. Use the Journal and Publication Information above to find the full article.

MeSH headings (categories)

This article was linked to the MESH Headings shown below.

5'-Guanylic Acid - analogs & derivatives
Adenosine Diphosphate
Adenosine Monophosphate
Adenylate Cyclase - chemistry
Animals
Binding Sites
Binding, Competitive
Catalytic Domain

Cattle
Guanosine Triphosphate
Guanylate Cyclase - chemistry; metabolism
Lung - enzymology
Nucleotides - metabolism
Solubility
Structural Homology, Protein
Substrate Specificity

Associated Chemicals: Nucleotides (0) ; Adenosine Diphosphate (58-64-0) ; Adenosine Monophosphate (61-19-8) ; 5'-Guanylic Acid (85-32-5) ; Guanosine Triphosphate (86-01-1) ; Adenylate Cyclase (EC 4.6.1.1) ; Guanylate Cyclase (EC 4.6.1.2)

These are the highest related articles currently in the database:

See 100+ related articles.