A kinetic study of interactions of (Rp)- and (Sp)-adenosine cyclic 3',5'-phosphorothioates with type II bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase.
A kinetic study of interactions of (Rp)- and (Sp)-adenosine cyclic 3',5'-phosphorothioates with type II bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase.
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(Rp)-和(Sp)-腺苷环3,5-硫代磷酸酯与II型牛心肌腺苷环3,5-磷酸依赖性蛋白激酶相互作用的动力学研究。
DOI：
10.1021/bi00261a028
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发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Kaiser,ET
中科院分区：
文献类型：
--
作者：
O'Brian,CA;Roczniak,SO;Bramson,HN;Baraniak,J;Stec,WJ;Kaiser,ET
S '。通过检查（RP）-和（Sp）-腺苷环3 '，5'-硫代磷酸（cAMPS）与这些位点的相互作用，研究了II型牛心肌cAMP依赖性蛋白激酶调节亚基上的S '-磷酸（cAMP）结合位点。虽然这两种非对映异构体的全酶的激活和结合到II型激酶的调节亚基，但cAMPS异构体与酶的相互作用之间存在显着差异。特别是，SP异构体比RP物质更有效，不仅在激活重建，以及直接分离，全酶，而且在抑制[3H] cAMP结合的调节亚基。存在SP异构体与调节亚基中的位点2结合的显著偏好。氢键上的调节subunitwith优先取向的环外氧，而不是硫代磷酰基残基的硫的官能团可能参与所观察到的选择性cAMPS的结合和激活。除了我们对cAMPS与cAMP依赖性蛋白激酶结合的立体选择性的发现外，我们还建立了一种从纯化的亚基重建全酶而不使调节蛋白变性的方法。牛心肌的cAMP依赖性蛋白激酶被认为以两种无活性的四聚体形式存在，每种形式含有两个相同的催化亚基和两个I型或两个II型调节亚基（Corbin等人，1978; Erlichmann等人，1973年; Bechtel & Beavo，1974年; Beavo等人，1974年）。自磷酸化位点位于II型调节二聚体中，但不在I型调节二聚体中（Corbin等人，1974年，1975年）。据信cAMP 1与调节亚基的结合导致全酶解离成两个活性催化亚基和调节亚基的二聚体（Corbin等人，1978年）。已经确定，每个II型调节单体具有两个不同的cAMP结合位点（Rannels和Corbin，1980），并且足以活化的结合的化学计量似乎是每个全酶两个cAMP分子（Kerlavage和Taylor，1982）。
S'. S'-phosphate (cAMP) binding sites on the regulatory subunit of the type II bovine cardiac muscle cAMP-dependent protein kinase was investigated by examining the interactions of (RP)-and (Sp)-adenosine cyclic 3', 5'-phosphorothioates (cAMPS) with these sites. While activation of the holoenzyme and binding to the regulatory subunit of the type II kinase were observed for both of these diastereomers, there were significant differences between the interactions of the cAMPS isomers with the enzyme. In particular, the SP isomer is more potent than the RP species not only in the activation of reconstituted, as well as directly isolated, holoenzyme but also in the in-hibition of [3H] cAMP binding to the regulatory subunit. A marked preference for the binding of the SP isomer to site 2 in the regulatory subunit exists. Hydrogen bonding of a functional group on the regulatory subunitwith preferential orientation toward the exocyclic oxygen rather than the sulfur of the thiophosphoryl residue may be involved in the observed selectivity of cAMPS binding and activation. In addition to our findings on the stereoselectivity of the binding of cAMPS to cAMP-dependent protein kinase, we have established a method for the reconstitution of holoenzyme from the purified subunits without subjecting the regulatory protein to dena-turing conditions. e cAMP-dependent protein kinase of bovine cardiac muscle is thought to occur in two inactive, tetrameric forms, each containing two identical catalytic subunits and two type I or two type II regulatorysubunits (Corbin et al., 1978; Erlichmann et al., 1973; Bechtel & Beavo, 1974; Beavo et al., 1974). Autophosphorylation sites existin the type II but not in the type I regulatory dimer (Corbin et al., 1974, 1975). The binding of cAMP1 to the regulatory subunits is believed to cause the holoenzyme to dissociate into two active catalytic subunits and a dimer of regulatory subunits (Corbin et al., 1978). It has been determined that each type II regulatory monomer has two nonidentical cAMP binding sites (Rannels & Corbin, 1980), and the stoichiometry of binding sufficient for activation appears to be two cAMP molecules per holoenzyme (Kerlavage & Taylor, 1982).