MECHANISM OF CAMP DEPENDENT PROTEIN KINASE 16.

环腺苷酸依赖蛋白的整体动力学机制 来自牛心脏的激酶将使用多种 不同的技术。 在没有 抑制剂和存在产物和死端抑制剂时 将在MgADP磷酸化的方向上进行。 由于从这些研究中获得的结果很少是结论性的， 上述实验将使用同位素交换进行补充 MgATP/MgADP、MgATP/磷酸肽和 肽/磷酸肽交换反应。 同位素分配 用3 H-肽、3 H-ADP和32 P-磷酸肽的实验将 也可用于证实动力学机制。 最后，同位素 β-非桥连180-ADP交换到桥连位置，如果 它的发生将作为磷酸肽的功能进行， 确定磷酸肽和MgADP的释放是否是随机的。 关于利率限制的位置和金额的信息 确定步骤将从上述所有内容中确定 实验方法与获得的额外信息， 同位素效应研究 有关化学机制的信息将通过以下方式获得： 完成MgADP磷酸化方向的pH研究 为了阐明酸基化学，并通过使用重原子 同位素效应，使用远程标记技术。 肽 含有β-180-丝氨酸（160缺失）和15 N-甘氨酸 耗尽14 N和β-160-丝氨酸（耗尽180）和14 N-丝氨酸（耗尽180）。 将制备甘氨酸（耗尽15 N），混合至天然甘氨酸中。 甘氨酸氮的丰度，并用于获得180 同位素效应，15 N作为报告子。 多个备用 将测试肽底物、核苷酸和条件， 寻找限速磷酰基转移的条件。

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.