The catalytic mechanism of mammalian adenylyl cyclase - Equilibrium binding and kinetic analysis of P-site inhibition
The catalytic mechanism of mammalian adenylyl cyclase - Equilibrium binding and kinetic analysis of P-site inhibition
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DOI：
10.1074/jbc.272.44.27787
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发表时间：
1997-10-31
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;Gilman, AG
通过使用P位点抑制剂2 '-[H-3]脱氧腺苷的平衡结合测量以及正向和反向反应（即分别为环状BMP和ATP合成）的动力学分析，探索了腺苷酸环化酶P位点抑制的机制。每个C-1/C-2异二聚体有一个2 '-脱氧腺苷结合位点; Kd为40 +/- 3 μ M。该发现仅在腺苷酸环化酶反应产物之一焦磷酸（PPi）存在下观察到。底物类似物Ap（CH 2）pp（α，β-亚甲基腺苷5 '-三磷酸）和环AMP在PPi存在下竞争P-位点，但P-位点类似物不竞争底物结合（在不存在PPi的情况下）。动力学分析表明，产物从酶中的释放是随机的。这些事实允许制定一个模型的腺苷酸环化酶反应，我们提供了大量的动力学支持。我们建议P-位点类似物作为产品释放的死端抑制剂，通过在活性位点结合来稳定酶-产品（E-PPi）复合物。虽然产物释放是随机的，但环AMP优先从酶解离。PPi的释放是缓慢的并且部分限速。
The mechanism of P-site inhibition of adenylyl cyclase has been probed by equilibrium binding measurements using 2'-[H-3]deoxyadenosine, a P-site inhibitor, and by kinetic analysis of both the forward and reverse reactions (i.e. cyclic BMP and ATP synthesis, respectively). There is one binding site for 2'-deoxyadenosine per C-1/C-2 heterodimer; the K-d is 40 +/- 3 mu M. finding 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.