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 (Asp103) 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.
