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J Biol Chem. 2015 Jun 19;290(25):15570-80. doi: 10.1074/jbc.M115.641431. Epub 2015 Apr 23.

The Influence of Nitric Oxide on Soluble Guanylate Cyclase Regulation by Nucleotides: ROLE OF THE PSEUDOSYMMETRIC SITE.

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From the Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037 and.
the Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CG Utrecht, The Netherlands.
From the Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037 and


Activation of soluble guanylate cyclase (sGC) by the signaling molecule nitric oxide (NO) leads to formation of the second messenger cGMP, which mediates numerous physiological processes. NO activates sGC by binding to the ferrous heme cofactor; the relative amount of NO with respect to sGC heme affects the enzyme activity. ATP can also influence the activity by binding to an allosteric site, most likely the pseudosymmetric site located in the catalytic domain. Here, the role of the pseudosymmetric site on nucleotide regulation was investigated by point mutations at this site. ATP inhibition kinetics of wild type and a pseudosymmetric site (α1-C594A/β1-D477A) variant of sGC was determined at various levels of NO. Results obtained show that in the presence of less than 1 eq of NO, there appears to be less than complete activation and little change in the nucleotide binding parameters. The most dramatic effects are observed for the addition of excess NO, which results in an increase in the affinity of GTP at the catalytic site and full activation of sGC. The pseudosymmetric site mutation only affected nucleotide affinities in the presence of excess NO; there was a decrease in the affinity for ATP in both the allosteric and catalytic sites. These observations led to a new kinetic model for sGC activity in the presence of excess NO. This model revealed that the active and allosteric sites show cooperativity. This new comprehensive model gives a more accurate description of sGC regulation by NO and nucleotides in vivo.


nitric oxide, guanylate cyclase (guanylyl cyclase), cyclic GMP (cGMP), ATP, signal transduction, heme

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