Dessauer C W, Gilman A G
Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
J Biol Chem. 1997 Oct 31;272(44):27787-95. doi: 10.1074/jbc.272.44.27787.
The mechanism of P-site inhibition of adenylyl cyclase has been probed by equilibrium binding measurements using 2'-[3H]deoxyadenosine, a P-site inhibitor, and by kinetic analysis of both the forward and reverse reactions (i.e. cyclic AMP and ATP synthesis, respectively). There is one binding site for 2'-deoxyadenosine per C1/C2 heterodimer; the Kd is 40 +/- 3 microM. Binding is observed only in the presence of one of the products of the adenylyl cyclase reaction, pyrophosphate (PPi). A substrate analog, Ap(CH2)pp (alpha,beta-methylene adenosine 5'-triphosphate), and cyclic AMP compete for the P-site in the presence of PPi, but P-site analogs do not compete for substrate binding (in the absence of PPi). Kinetic analysis indicates that release of products from the enzyme is random. These facts permit formulation of a model for the adenylyl cyclase reaction, for which we provide substantial kinetic support. We propose that P-site analogs act as dead-end inhibitors of product release, stabilizing an enzyme-product (E-PPi) complex by binding at the active site. Although product release is random, cyclic AMP dissociates from the enzyme preferentially. Release of PPi is slow and partially rate-limiting.
已通过使用2'-[3H]脱氧腺苷（一种P位点抑制剂）进行平衡结合测量，以及对正向和反向反应（即分别为环磷酸腺苷和三磷酸腺苷合成）进行动力学分析，来探究腺苷酸环化酶P位点抑制的机制。每个C1/C2异二聚体有一个2'-脱氧腺苷结合位点；解离常数（Kd）为40±3微摩尔。仅在腺苷酸环化酶反应的一种产物焦磷酸（PPi）存在的情况下才能观察到结合。在PPi存在的情况下，底物类似物Ap(CH2)pp（α,β-亚甲基腺苷5'-三磷酸）和环磷酸腺苷竞争P位点，但P位点类似物不竞争底物结合（在没有PPi的情况下）。动力学分析表明产物从酶上的释放是随机的。这些事实使得能够构建一个腺苷酸环化酶反应模型，我们为此提供了大量的动力学支持。我们提出P位点类似物作为产物释放的终产物抑制剂，通过在活性位点结合来稳定酶-产物（E-PPi）复合物。尽管产物释放是随机的，但环磷酸腺苷优先从酶上解离。PPi的释放缓慢且部分限速。
