Atomic level mutagenesis to study hydrogen bonds

描述（由申请人提供）： 了解酶如何实现其巨大的催化能力和精致的特异性是生物功能研究的核心。在过去的几十年里，酶的功能和行为的许多基本特征已经阐明。然而，酶的非凡的速率增强和特异性不能定量地解释。非天然氨基酸被要求在一个前所未有的深度探测什么是可以说是酶和非催化溶液反应之间的最深刻的区别，而不是水溶液中的特异性和高度专业化的酶活性位点进行反应的能量后果。将产生一系列的甾酮异构酶的变体，其中的活性位点的天冬氨酸残基（天冬氨酸103），捐赠的氢键的性质是不同的系统和合理的。这些研究将加深我们对酶活性位点内的氢键能量学的理解，并推进我们对酶环境的性质的理解，这些性质决定了这种氢键的性质和能量学。这些实验将为在酶的研究中系统而深入地应用化学微扰法奠定基础。

DESCRIPTION (provided by applicant): Understanding how enzymes achieve their enormous catalytic power and exquisite specificity is central to the study of biological function. Over the past decades many basic features of enzyme function and behavior have been illuminated. Nevertheless, the extraordinary rate enhancements and specificites of enzymes cannot be accounted for quantitatively. Unnatural amino acids are required to probe at an unprecedented depth the energetic consequences of what is arguably the most profound difference between enzymatic and uncatalyzed solution reactions-carrying out reactions in the idiosyncratic and highly specialized enzyme active site instead of aqueous solution. A series of ketosteroid isomerase variants will be generated in which the properties of an active site aspartic acid residue (Asp 103) that donates a hydrogen bond are varied systematically and rationally. These studies will deepen our understanding of hydrogen bonding energetics within an enzyme active site and advance our understanding of the properties of the enzymatic environment that dictate the nature and energetics of such hydrogen bonds. These experiments will serve as a foundation for the use of systematic and incisive chemical perturbation in the study of enzymes.