Ruiz-Stewart Inez, Kazerounian Shiva, Pitari Giovanni M, Schulz Stephanie, Waldman Scott A
Division of Clinical Pharmacology, Department of Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Eur J Biochem. 2002 Apr;269(8):2186-93. doi: 10.1046/j.1432-1033.2002.02874.x.
Nitric oxide (NO), the principal endogenous ligand for soluble guanylate cyclase (sGC), stimulates that enzyme and accumulation of intracellular cGMP, which mediates many of the (patho) physiological effects of NO. Previous studies demonstrated that 2-substituted adenine nucleotides, including 2-methylthioATP (2MeSATP) and 2-chloroATP (2ClATP), allosterically inhibit guanylate cyclase C, the membrane-bound receptor for the Escherichia coli heat-stable enterotoxin in the intestine. The present study examined the effects of 2-substituted adenine nucleotides on crude and purified sGC. 2-Substituted nucleotides inhibited basal and NO-activated crude and purified sGC, when Mg2+ served as the substrate cation cofactor. Similarly, 2-substituted adenine nucleotides inhibited those enzymes when Mn2+, which activates sGC in a ligand-independent fashion, served as the substrate cation cofactor. Inhibition of sGC by 2-substituted nucleotides was associated with a decrease in Vmax, consistent with a noncompetitive mechanism. In contrast to guanylate cyclase C, 2-substituted nucleotides inhibited sGC by a guanine nucleotide-independent mechanism. These studies demonstrate that 2-substituted adenine nucleotides allosterically inhibit basal and ligand-stimulated sGC. They support the suggestion that allosteric inhibition by adenine nucleotides is a general characteristic of the family of guanylate cyclases. This allosteric inhibition is mediated by direct interaction of adenine nucleotides with sGC, likely at the catalytic domain in a region outside the substrate-binding site.
一氧化氮（NO）是可溶性鸟苷酸环化酶（sGC）的主要内源性配体，可刺激该酶并促进细胞内cGMP的积累，而cGMP介导了NO的许多（病理）生理效应。先前的研究表明，2-取代的腺嘌呤核苷酸，包括2-甲硫基ATP（2MeSATP）和2-氯ATP（2ClATP），可别构抑制鸟苷酸环化酶C，后者是肠道中大肠杆菌热稳定肠毒素的膜结合受体。本研究考察了2-取代的腺嘌呤核苷酸对粗制和纯化的sGC的影响。当Mg2+作为底物阳离子辅因子时，2-取代的核苷酸可抑制基础状态及NO激活的粗制和纯化的sGC。同样，当以不依赖配体的方式激活sGC的Mn2+作为底物阳离子辅因子时，2-取代的腺嘌呤核苷酸也可抑制这些酶。2-取代的核苷酸对sGC的抑制作用与Vmax降低有关，这与非竞争性机制一致。与鸟苷酸环化酶C不同，2-取代的核苷酸通过不依赖鸟嘌呤核苷酸的机制抑制sGC。这些研究表明，2-取代的腺嘌呤核苷酸可别构抑制基础状态及配体刺激的sGC。它们支持了腺嘌呤核苷酸的别构抑制是鸟苷酸环化酶家族的一个普遍特征这一观点。这种别构抑制是由腺嘌呤核苷酸与sGC的直接相互作用介导的，可能发生在底物结合位点之外区域的催化结构域。
