Dynamic Analysis of Enzyme Functions

该研究项目特别关注酶对底物的分子识别等方面的研究。通过对10种酶的平衡和动力学分析，利用吸收光谱、荧光光谱和核磁共振（NMR）进行了研究。结果令人满意。本文介绍了一种微型断流装置和一种温度跃变装置，前者仅用十分之一体积的样品就能达到与常规装置相同的性能，后者则能用快速温度交换装置在较大的温差范围内使温度跳升或跳降。N-溴代丁二酰亚胺快速化学修饰动力学分析可用于研究酶反应位点色氨酸残基的状态。根据核磁共振氢氘交换的研究结果，讨论了蛋白质分子的涨落。所有这些酶系统的快速动力学分析表明，它的普遍性，酶和底物的结合进行至少两个步骤。在这个两步机制中，形成松散结合中间体的双分子过程之后是单分子异构化过程，导致形成特定的强结合复合物。酶分子的内部波动被认为在第二个过程中起着重要作用。

This research project has been focused particularly on the study of molecular recognition of substrate by enzymes among many other aspects. The study was conducted by means of absorption spectroscopy, fluorescence spectroscopy, and nuclear magnetic resonance (NMR) through equilibrium and kinetic analyses with about 10 kinds of enzymes. Satisfactory results were obtained. A micro-stopped flow apparatus which, as compared with the conventional apparatus, exhibits the equal degree of performance with only one tenth volume of the sample, and a temperaturejump apparatus which enables one to change the temperature either jumping-up or-down over a relatively wide defference by using a rapid-temperature exchange device, were constructed. Kinetic analysis of rapid chemical modification by Nbromosuccinimide was proved to be useful for investigating the state of tryptophan residues at the reactive site of enzyme. Flucuation of a protein molecule was discussed according to the results of NMR study on hydrogen-deuterium exchange. Rapid-kinetic analyses of all those enzyme systems suggested it universal that binding of an enzyme and the substrate proceeds with at least two steps. In this two step mechanism a bi-molecular process to form a loosely bound intermediate is followed by a uni-molecular isomerization process which results in the formation of a specific, strongly-bound complex. Internal fluctuation of the enzyme molecule is believed to play an important role in the second process.