A functional domain of the α1 subunit of soluble guanylyl cyclase is necessary for activation of the enzyme by nitric oxide and YC-1 but is not involved in heme binding
A functional domain of the α1 subunit of soluble guanylyl cyclase is necessary for activation of the enzyme by nitric oxide and YC-1 but is not involved in heme binding
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DOI：
10.1074/jbc.m212740200
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发表时间：
2003-04-04
影响因子：
4.8
通讯作者：
Behrends, S
中科院分区：
文献类型：
--
作者：
Koglin, M;Behrends, S
可溶性鸟苷酸环化酶是由α（1）和β（1）亚基组成的异二聚体酶，并且是内源性一氧化氮和鸟苷酸环化酶调节剂YC-1的重要靶标。这两种物质对酶的激活都依赖于血红素辅基的存在。目前还不清楚这个血红素修复基团是否夹在α（1）和β（1）亚基之间，或者它是否只与β（1）亚基结合。在这里，我们分析了进行性氨基末端缺失突变体的人α（1）亚基共表达后，与人β（1）亚基在杆状病毒/Sf 9系统。光谱、生物化学和药理学分析表明，α（1）亚基的前259个氨基酸可以被删除，而不会丧失对一氧化氮（NO）或YC-1的敏感性，也不会丧失相应酶复合物与β（1）亚基的血红素结合。这与以前的数据相反，表明NO敏感性和功能性血红素结合位点需要牛α（1）和β（1）亚基的全长氨基末端。α（1）亚基的前364个氨基酸的进一步缺失导致具有保留的血红素结合的酶复合物，但是丧失对NO或YC-1的敏感性，尽管通过NO结合至血红素辅基诱导典型的光谱位移。我们的结论是：1）α（1）亚基的氨基末端部分不参与血红素结合，2）α（1）亚基的氨基酸259-364代表了NO激活信号转导的重要功能结构域，并可能代表了NO致敏物质如YC-1的靶点。
Soluble guanylyl cyclase is a heterodimeric enzyme consisting of an alpha(1) and a beta(1) subunit and is an important target for endogenous nitric oxide and the guanylyl cyclase modulator YC-1. The activation of the enzyme by both substances is dependent on the presence of a prosthetic heme group. It has been unclear whether this prosthetic heme group is sandwiched between the alpha(1) and beta(1) subunits or whether it exclusively binds to the beta(1) subunit. Here we analyze progressive amino-terminal deletion mutants of the human alpha(1) subunit after co-expression with the human beta(1) subunit in the baculovirus/Sf9 system. Spectral, biochemical, and pharmacological analysis shows that the first 259 amino acids of the alpha(1) subunit can be deleted without loss of sensitivity to nitric oxide (NO) or YC-1 or loss of heme binding of the respective enzyme complex with the beta(1) subunit. This is in contrast to previous data indicating that NO sensitivity and a functional heme binding site requires full-length amino termini of bovine alpha(1) and beta(1) subunits. Further deletion of the first 364 amino acids of the alpha(1) subunit leads to an enzyme complex with preserved heme binding but loss of sensitivity to NO or YC-1 despite induction of the typical spectral shift by NO binding to the prosthetic heme group. We conclude that 1) the amino-terminal part of the alpha(1) subunit is not involved in heme binding and 2) amino acids 259-364 of the alpha(1) subunit represent an important functional domain for the transduction of the NO activation signal and likely represent the target for NO-sensitizing substances like YC-1.