MECHANISMS OF ENZYMIC AND HYDRIDE TRANSFERS

描述(由申请人提供)： 该项目的目标是利用X射线结晶学、计算机模拟、定点突变和动力学测量来确定酶活性部位的哪些结构特征有助于有效催化氢离子转移。氢的转移，通常是以质子或氢化物离子的形式，是生物学中最常见的化学反应，也可能是最简单的，但这并不总是一个快速进行的容易的反应。当给体和受体原子是未活化的碳或氧原子时，非催化反应非常缓慢，因为-C-H和-O-H基团不是很酸性(通常pKa值都在20附近)。然而，在某些情况下，酶能够加速H+和H-从-C-H和-O-H的转移，达到每秒超过1000个数量级的周转次数。确定酶如何激活底物、辅因子和它们自己的催化基团来实现这种催化是本项目的目标。我们选择了几个模型系统进行研究。对于质子转移催化：突变酶(Galm)，它催化开环和质子转移；以及酮类异构酶(KSI)，它利用典型的酸/碱机制。氢化物转移：木糖异构酶(XyI)，它催化糖环的打开，然后是金属介导的1，2-氢化物转移和二氢叶酸还原酶(DHFR)，其中氢化物供体是NAD。对于变形酶和KSI，我们的目的是了解这些酶是如何增加催化碱的碱性和降低碳酸的pKa的，以及过渡态是如何稳定的。对于XYI，我们的目标是了解活性中心的两个金属离子如何合作促进氢化物转移，以及开环反应是如何被催化的。对于DHFR，我们正在研究氢化物供体和底物定位的方向和距离要求，以及辅酶和底物菌株的可能作用。所有这些酶的活性中心的质子化环境和蛋白质动力学在催化中的作用也正在通过计算和超高分辨率(超过约IA分辨率)的X射线衍射来探索。

DESCRIPTION (provided by applicant): The objective of this project is to use X-ray crystallography, computer simulations, site-directed mutagenesis and kinetic measurements to determine what structural features in an enzyme active site promote efficient catalysis of hydrogen ion transfer. Transfer of a hydrogen, usually in the form of either a proton or a hydride ion is the most common chemical reaction in biology, and is also perhaps the simplest, yet it is not always an easy reaction to carry out rapidly. When the donor and acceptor atoms are unactivated carbons or oxygens, the uncatalyzed reactions are very slow, because the -C-H and -O-H groups are not very acidic (both typically have pKa values near 20). Yet enzymes are able to accelerate the transfer of H+ and H- from -C-H and -O-H by more than 15 orders of magnitude in some cases, achieving turnover numbers exceeding 1000 per second. Establishing how enzymes activate substrates, cofactors and their own catalytic groups to effect such catalysis is the goal of this project. We have selected several model systems for study. For proton transfer catalysis: mutarotase (GalM), which catalyses both ring opening and proton transfer; and ketosteroid isomerase (KSI), which utilizes a prototypical acid/base mechanism. For hydride transfer: xylose isomerase (XyI), which catalyzes sugar ring opening followed by metal-mediated 1,2-hydride transfer and dihydrofolate reductase (DHFR), where the hydride donor is NAD. For mutarotase and KSI our aim is to understand how the enzymes increase the basicity of the catalytic base and lower the pKa of the carbon acid and how the transition states are stabilized. For XyI, we aim to learn how the two metal ions in the active site cooperate to promote hydride transfer and how the ring-opening reaction is catalyzed. For DHFR we are studying the orientation and distance requirements for hydride donor and substrate positioning, and the possible role of coenzyme and substrate strain. The protic environment in the active sites and the role of protein dynamics in catalysis are also being probed for all these enzymes computationally and by ultra-high resolution (beyond approximately IA resolution) X-ray diffraction.