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J. Biol. Chem., Vol. 281, Issue 31, 21763-21770, August 4, 2006

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Functional Characterization of Two Nucleotide-binding Sites in Soluble Guanylate Cyclase^*

Shinsuke Yazawa, Hidemi Tsuchiya, Hiroshi Hori, and Ryu Makino¹

From the Department of Life Science, College of Science, Rikkyo University, Nishi-ikebukuro 3-34-1, Toshima-ku, Tokyo 171-8501 and the Division of Bioengineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan

Soluble guanylate cyclase is a heterodimeric hemoprotein composed of - and -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 ,-methylene guanosine 5'-triphosphate (GMP-CPP). In contrast, 2'-dADP occupied both sites with equivalent affinities. Adenosine-5'-,-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.

Received for publication, August 15, 2005 , and in revised form, June 1, 2006.

^* This work was supported by Frontier Project "Adaptation and Evolution of Extremophile" of Rikkyo University and Grants-in-Aid from the Ministry of Culture, Education, Sports, Science and Technology of Japan (to R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed: Dept. of Life Science, and Frontier Project "Adaptation and Evolution of Extremophile," College of Science, Rikkyo (St. Paul's) University, Nishi-ikebukuro 3-34-1, Toshima-ku, Tokyo 171-8501, Japan. Fax: 81-3-3985-2386; E-mail: rmakino{at}rikkyo.ne.jp.

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