Dessauer C W, Scully T T, Gilman A G
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
J Biol Chem. 1997 Aug 29;272(35):22272-7. doi: 10.1074/jbc.272.35.22272.
Fragments of the two cytoplasmic domains of mammalian adenylyl cyclases can be synthesized independently (and abundantly) as soluble proteins; Gsalpha- 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 Gsalpha, 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 Kd (0.1 microM) corresponds to the EC50 for enzyme activation. The affinity of forskolin for adenylyl cyclase is greatly reduced in the absence of Gsalpha ( approximately 40 microM). 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 Km for ATP or the Ki for Ap(CH2)pp. It thus seems unlikely that this region is part of the active site. However, a mutation in the C1 domain (E518A) causes 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.
哺乳动物腺苷酸环化酶的两个细胞质结构域片段可以独立（且大量）合成成为可溶性蛋白质；将它们混合后，可恢复由Gsα和福斯高林刺激的酶活性。我们利用该系统来表征腺苷酸环化酶与福斯高林及其底物ATP之间的相互作用。在存在Gsα的情况下，仅占据一个福斯高林结合位点就能激活腺苷酸环化酶。通过平衡透析鉴定出福斯高林的单个结合位点；其解离常数（Kd，0.1微摩尔）对应于酶激活的半数有效浓度（EC50）。在不存在Gsα的情况下（约40微摩尔），福斯高林对腺苷酸环化酶的亲和力大大降低。未检测到福斯高林与该酶的单个细胞质结构域的结合。通过平衡透析还检测到ATP类似物α,β-亚甲基ATP（Ap(CH2)pp）的单个结合位点。在单个结构域中未观察到这种结合。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末端附近的一个修饰的P环序列与ATP结合有关。该区域内保守的非甘氨酸残基发生突变，对ATP的米氏常数（Km）或Ap(CH2)pp的抑制常数（Ki）没有显著影响。因此，该区域似乎不太可能是活性位点的一部分。然而，C1结构域中的一个突变（E518A）导致对Ap(CH2)pp的结合亲和力下降10倍。该残基和酶的活性位点可能位于两个胞质结构域之间的界面处。
