Interactions of forskolin and ATP with the cytosolic domains of mammalian adenylyl cyclase
Interactions of forskolin and ATP with the cytosolic domains of mammalian adenylyl cyclase
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DOI：
10.1074/jbc.272.35.22272
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发表时间：
1997-08-29
影响因子：
4.8
通讯作者：
Gilman, AG
中科院分区：
文献类型：
--
作者：
Dessauer, CW;Scully, TT;Gilman, AG
哺乳动物腺苷酸环化酶的两个细胞质结构域的片段可以作为可溶性蛋白独立（和大量）合成；G(s α)-和福斯克林刺激的酶活性在它们的混合物中恢复。我们利用这个系统来表征腺苷酸环化酶与福斯克林及其底物ATP的相互作用。在G（s α）存在的情况下，腺苷酸环化酶仅在占据一个福斯克林结合位点时被激活，通过平衡透析确定了福斯克林的单一结合位点；其K-d （0.1 μ M）对应于酶激活的EC50，在G（s α）缺失的情况下，福斯克林对腺苷酸环化酶的亲和力大大降低（与40 μ M相似）。没有检测到forskolin与酶的单个细胞质结构域的结合。平衡透析还检测到ATP类似物α， β -亚甲基ATP （Ap(CH2)pp）的单一结合位点，但在单个结构域上没有观察到这种结合，Ap(CH2)pp的结合不受腺苷酸环化酶p位点抑制剂的影响，位于腺苷酸环化酶羧基端附近的修饰p环序列与ATP的结合有关。该区域内的非甘氨酸残基对ATP的K-m和Ap(CH2)pp的K-i没有显著变化。因此，该区域似乎不太可能是活性位点的一部分，然而，C-1结构域（E518A）的突变导致Ap(CH2)pp的结合亲和力降低10倍，该残基和酶的活性位点可能位于两个细胞质结构域之间的界面。
Fragments of the two cytoplasmic domains of mammalian adenylyl cyclases can be synthesized independently (and abundantly) as soluble proteins; G(s alpha)- and forskolin-stimulated enzymatic activity is restored upon their mixture. We have utilized this system to characterize the interactions of adenylyl cyclase with forskolin and its substrate, ATP, In the presence of G(s alpha), adenylyl cyclase is activated in response to occupation of only one forskolin-binding site, A single binding site for forskolin was identified by equilibrium dialysis; its K-d (0.1 mu M) corresponds to the EC50 for enzyme activation, The affinity of forskolin for adenylyl cyclase is greatly reduced in the absence of G(s alpha) (similar to 40 mu M). Binding of forskolin to the individual cytoplasmic domains of the enzyme was not detected. A single binding site for the ATP analog, alpha,beta-methylene ATP (Ap(CH2)pp), was also detected by equilibrium dialysis, Such binding was not observed with the individual domains, Binding of Ap(CH2)pp was unaffected by P-site inhibitors of adenylyl cyclase, A modified P-loop sequence located near the carboxyl terminus of adenylyl cyclase has been implicated in ATP binding, Mutation of the conserved, non-glycine residues within this region caused no significant changes in the K-m for ATP or the K-i for Ap(CH2)pp. It thus seems unlikely that this region is part of the active site, However, a mutation in the C-1 domain (E518A) cause a 10-fold decrease in the binding affinity for Ap(CH2)pp, This residue and the active site of the enzyme may lie at the interface between the two cytosolic domains.