Havsteen B H
Biochem J. 1985 Dec 15;232(3):791-7. doi: 10.1042/bj2320791.
A general method has been devised for the exact evaluation of the rate constants of the elementary steps of a system consisting of any number of coupled reactions. The method is independent of the structure of the network of coupled steps. The precision of the data evaluation is solely dependent on the quality of the detection unit. The application of the method is illustrated with data collected for the binding of the competitive inhibitor proflavine to chymotrypsin under such conditions that five states of the enzyme are required to interpret the results. In the absence of a substance possessing the binding specificity, the enzyme is present as an equilibrium between an active and an inactive conformer. The latter prevails. The binding of the specific inhibitor releases a slow proton transfer from the medium to the alpha-amino group of Ile-16. Subsequently, the enzyme-inhibitor (or enzyme-substrate) complex re-arranges to the catalytically active form, which is retained until the supply of specific substrate is exhausted. The control features described are general, but are particularly conspicuous under the special environmental conditions used here. A comparison between data for alpha- and delta-forms of chymotrypsin showed that the chain ends of the former impeded the substrate binding and that the activity controlling conformational change occurred in the interior of the enzyme molecule.
已设计出一种通用方法，用于精确评估由任意数量的耦合反应组成的系统中基本步骤的速率常数。该方法与耦合步骤网络的结构无关。数据评估的精度仅取决于检测单元的质量。通过在需要用酶的五种状态来解释结果的条件下收集的关于竞争性抑制剂原黄素与胰凝乳蛋白酶结合的数据，说明了该方法的应用。在没有具有结合特异性的物质的情况下，酶以活性构象和非活性构象之间的平衡形式存在。后者占主导。特异性抑制剂的结合释放出一个缓慢的质子从介质转移到异亮氨酸-16的α-氨基上。随后，酶-抑制剂（或酶-底物）复合物重排为催化活性形式，该形式一直保持到特异性底物供应耗尽。所描述的控制特征是普遍的，但在这里使用的特殊环境条件下尤为明显。对胰凝乳蛋白酶α-型和δ-型数据的比较表明，前者的链端阻碍底物结合，且活性控制构象变化发生在酶分子内部。
