Interactions of Soluble Guanylate Cyclase with a P-Site Inhibitor: Effects of Gaseous Heme Ligands, Azide, and Allosteric Activators on the Binding of 2鈥?Deoxy-3鈥?GMP

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• 作者：Ryu Makino ; Shinsuke Yazawa ; Hiroshi Hori ; Yoshitsugu Shiro
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：November 20, 2012
• 年：2012
• 卷：51
• 期：46
• 页码：9277-9289
• 全文大小：741K
• 年卷期：v.51,no.46(November 20, 2012)
• ISSN：1520-4995

文摘

Nitric oxide (NO) elicits a wide variety of physiological responses by binding to the heme in soluble guanylate cyclase (sGC) to stimulate cGMP production. Although nucleotides, such as ATP or GTP analogues, have been reported to regulate the signaling of NO binding from the heme site to the catalytic site, the other regulatory functions of nucleotides remain unexamined. Among the nucleotides tested, we found that 2鈥?d-3鈥?GMP acted as a potent noncompetitive inhibitor with respect to Mn-GTP, when the ferrous enzyme combined with NO, CO, or allosteric activator BAY 41-2272. 2鈥?d-3鈥?GMP also displayed nearly identical patterns of inhibition for the ferric enzyme, in which the binding of N₃^{鈥?/sup> or BAY 41-2272 significantly increased the inhibitory effects of the nucleotide. Equilibrium dialysis measurements using the CO-ligated enzyme in the presence of allosteric activators demonstrated that 2鈥?d-3鈥?GMP exclusively binds to the catalytic site of sGC. Furthermore, the affinity of 2鈥?d-3鈥?GMP for the enzyme was found to increase upon addition of foscarnet, an analogue of PP_i. These findings together with other kinetic results imply that 2鈥?d-3鈥?GMP acts as a P-site inhibitor probably by forming a dead-end complex, sGC鈥?鈥?d-3鈥?GMP鈥揚P_i, in the catalytic reaction. The formation of the complex of the enzyme with 2鈥?d-3鈥?GMP does not seem to be associated with changes in the Fe鈥損roximal His bond strength, because the CO coordination state or the redox potentials of the enzyme-heme complex are virtually unaffected.}