Houge G, Steinberg R A, Ogreid D, Døskeland S O
Department of Anatomy, University of Bergen, Norway.
J Biol Chem. 1990 Nov 15;265(32):19507-16.
To probe the functional significance of the two cAMP-binding sites (A and B) on each regulatory subunit (RI) of cAMP-dependent protein kinase I, the dissociation of cAMP was studied from wild type RI liganded on site A, site B, or both sites, in the absence and presence of catalytic subunit (C). C enhanced the dissociation of cAMP from RI monoliganded on site A or B more than from A,B-biliganded RI, the rate difference being several orders of magnitude in the absence of Mg/ATP and about 7-fold in the presence of Mg/ATP. The catalytically active site of C was involved, since substrates or pseudosubstrates completely and competitively inhibited the action of C in the absence or presence of Mg/ATP. There was no evidence that C, by binding to one monomer of the RI dimer, affected the binding of cAMP to the other monomer. Likewise, there was no evidence for stable complexes of C and cAMP bound to the same R monomer. C enhanced the dissociation of cAMP from R subunits mutated in site A (RIGlu200, which is mutant RI in which glycine 200 is replaced by glutamic acid) or site B (RITrp334, which is mutant RI in which arginine 334 is replaced by tryptophan) to the same extent as from wild type RI monoliganded with cAMP. This indicates that the properties of nonmutated cAMP-binding sites in RIGlu200 and RITrp334 are modulated in a normal manner by C. Mutant RI defective in site A (RIGlu200) had the same rate and equilibrium cAMP binding properties as did site B of RI with its A site unoccupied. This means that mutational inactivation of one cAMP-binding site of RI can occur without altering the other intrachain cAMP site. By all criteria tested, therefore, RIGlu200 appears to be a valid model for RI with a vacant or nonoccupiable site A. Cooperativity of cAMP binding to the two cAMP-binding sites (A and B) of RI was observed only in the presence of C, the apparent Hill coefficient of cAMP binding being about 2 in the presence of a constant, high concentration of free C. C did not induce cooperativity of cAMP binding to RIGlu200 but caused a dramatic decrease of the apparent cAMP affinity of RIGlu200 relative to wild type RI.
为了探究环磷酸腺苷（cAMP）依赖性蛋白激酶I每个调节亚基（RI）上两个cAMP结合位点（A和B）的功能意义，我们研究了在有无催化亚基（C）存在的情况下，cAMP从结合于位点A、位点B或两个位点的野生型RI上的解离情况。与结合于A、B双位点的RI相比，C增强了cAMP从单结合于位点A或位点B的RI上的解离，在不存在Mg/ATP时速率差异达几个数量级，在存在Mg/ATP时约为7倍。C的催化活性位点参与其中，因为底物或假底物在有无Mg/ATP的情况下均能完全竞争性抑制C的作用。没有证据表明C通过结合RI二聚体的一个单体而影响cAMP与另一个单体的结合。同样，也没有证据表明C和cAMP结合于同一个R单体形成稳定复合物。C增强了cAMP从位点A突变的R亚基（RIGlu200，即甘氨酸200被谷氨酸取代的突变型RI）或位点B突变的R亚基（RITrp334，即精氨酸334被色氨酸取代的突变型RI）上的解离，其程度与从单结合cAMP的野生型RI上的解离程度相同。这表明RIGlu200和RITrp334中未突变的cAMP结合位点的特性受到C的正常调节。位点A有缺陷的突变型RI（RIGlu200）与A位点未被占据的RI的位点B具有相同的cAMP结合速率和平衡特性。这意味着RI的一个cAMP结合位点的突变失活可以在不改变另一个链内cAMP位点的情况下发生。因此，根据所有测试标准，RIGlu200似乎是一个A位点空缺或不可占据的RI的有效模型。仅在存在C的情况下观察到cAMP与RI两个cAMP结合位点（A和B）结合的协同性，在存在恒定高浓度游离C的情况下cAMP结合的表观希尔系数约为2。C并未诱导cAMP与RIGlu200结合的协同性，但相对于野生型RI，导致RIGlu200的表观cAMP亲和力显著降低。
