MECHANISM OF CAMP DEPENDENT PROTEIN KINASE 16.

环磷酸腺苷依赖蛋白的整体动力学机制 来自牛心脏的激酶将使用各种不同的 不同的技术。初始速度研究中的缺失 抑制剂以及在产品和死胡同抑制剂存在的情况下 将朝着镁ADP的磷酸化方向进行。 由于从这些研究中获得的结果很少是决定性的， 上述实验将使用同位素交换进行补充。 镁-三磷酸腺苷/镁-腺苷二磷酸、镁-三磷酸腺苷/磷酸肽和 肽/磷酸肽交换反应。同位素分配 用~3H-肽、~3H-ADP和~(32)P-磷酸小肽进行实验将 也可用来佐证动力学机制。最后，同位素 将β-非桥联180-ADP交换到桥接位置，如果 它的发生将作为磷酸肽的函数来进行 确定磷酸肽和镁ADP的释放是否是随机的。 关于税率限制的地点和金额的信息 确定步骤将根据以上所有内容确定 从以下方面获得附加信息的实验方法 同位素效应研究。 有关化学机理的信息将通过以下方式获得 完成镁ADP磷酸化方向的pH研究 通过使用重原子来阐明酸基化学和 使用远程标记技术的同位素效果。肽类 含有β-180-丝氨酸(缺失160)和15N-甘氨酸 缺乏14N和β-160-丝氨酸(缺乏180)和14N- 甘氨酸(耗尽15N将被制备，混合到天然的 甘氨酸氮的丰度并用于获得180 以15N为报告分子的同位素效应。多个备选方案 将对多肽底物、核苷酸和条件进行测试 找出限制磷酰转移速率的条件。

The overall kinetic mechanism of cyclic AMP-dependent protein kinase from bovine heart will be determined using a variety of different techniques. Initial velocity studies in the absence of inhibitors and in the presence of product and dead-end inhibitors will be carried out in the direction of phosphorylation of MgADP. Since results obtained from these studies are rarely conclusive, the above experiments will be complemented using isotope exchange at equilibrium for the MgATP/MgADP, MgATP/ phosphopeptide and peptide/phosphopeptide exchange reactions. Isotope partitioning experiments with 3H-peptide, 3H-ADP and 32P-phosphopeptide will also be used to corroborate kinetic mechanism. Finally, isotope exchange of beta-nonbridged 180-ADP into the bridge position, if it occurs will be carried out as a function of phosphopeptide to determine whether release of phosphopeptide and MgADP is random. Information on the location and amount of limitation of rate determining steps will Be determined from all of the above experimental approaches with additional information obtained from isotope effect studies. Information on the chemical mechanism will be obtained by completing the pH studies in the direction of MgADP phosphorylation to elucidate acid-based chemistry and through the use of heavy atom isotope effects using the remote label technique. Peptides containing beta-180-serine (depleted of 160) and 15N-glycine depleted of 14N and beta-160-serine (depleted of 180) and 14N- glycine (depleted of 15N will be prepared, mixed to the natural abundance of the glycine nitrogen and used to obtain the 180 isotope effect with 15N as the reporter. A number of alternate peptide substrates, nucleotides and conditions will be tested to find conditions for rate limiting phosphoryl transfer.