Enzymes are the molecules within cells that make the chemical reactions necessary for life happen fast and happen correctly. These reactions govern all aspects of cellular processes, from the extraction of energy from food, to the building of structures needed for constructing cells and organs, to the exquisite control of these processes as an organism experiences different environments and stresses. This research aims to contribute to the fundamental understanding of enzyme action via investigation of a highly tractable, model enzyme, bacterial ketosteroid isomerase. This enzyme has several features that will allow its study at unprecedented depth and breadth. Specifically, this research entails a finely crafted blend of structural, dynamic and biochemical studies. Furthermore, this enzyme is tractable for chemical synthesis so that changes at the level of individual amino acids can be introduced systematically and thoroughly, without the limitations of the 20 naturally occurring amino acids. Most broadly, a central unanswered question in enzyme mechanism is the role of the protein structure surrounding the active site where catalysis takes place, and this research will provide incisive tests of the properties of the enzyme environment.
Understanding how enzymes function is fundamental for understanding life itself. Furthermore, the majority of drug targets are enzymes, so that a deeper fundamental understanding of the action of enzymes may allow researchers to better design potent inhibitors that can act as drugs for numerous disease states. A deep understanding of enzyme action may also provide the information needed to design new enzymes that catalyze new and beneficial reactions that can be used in the pharmaceutical and chemical industries.
The broader impact of this project involves the training of graduate students in rigorous, quantitative aspects of biological chemistry, a background that will allow these students to contribute in many ways in their individual careers, as teachers, in applied or fundamental research, and in evaluating policy from a scientific standpoint. Further, the basic nature of the research renders it possible for undergraduates to obtain meaningful research experiences. In particular there are plans to ensure the inclusion of underrepresented minorities in this research, an opportunity that may greatly help stimulate the inclusion of such students in the future in the scientific community.
Mechanistic Investigations of Ketosteroid Isomerase Enzymes catalyze, or speed, specific reactions carried out by all living things. Thus, they are at the core of biological function, and we desire a deep understanding of their function both because they are so fundamental to life and because their aberrant behavior often results in disease. Better understanding of how enyzmes work may lead to better ways to inhibit the action of enzymes from infectious agents, to control the activities of malfunctioning enzymes, and to design new enzymes with desirable health and economic benefits. We have chosen to study ketosteroid isomerase (KSI) from bacteria because it has features in common with many other enzymes –so that generality from our studies is a reasonable expectation- and it is extraordinarily tractable to many different types of experimental approaches. Often the deepest insights are obtained by looking at a problem from multiple vantage points. To understand catalysis, we and others consider the reaction species of the highest energy, the transition state, and how an enzyme might stabilize it –thereby increasing the probability or rate of reaction. Two general properties of reactants change as a reaction proceeds: where the electrons are –in other words, electrostatics, and the shape or geometry of the reactant. Because both change together, it has been very difficult to distinguish to what extent enzymes preferentially recognize electrostatic vs geometrical changes. Using compounds that are related to but distinct from the actual reactants and transition states, we were able to systematically change either the electrostatic properties or the geometric properties –in other words, untangling these two elements. The results of these studies suggested that KSI does less to stabilize electrostatic changes than had previously been assumed and that it is remarkable in its ability to distinguish geometrical changes, even on the scale of hundredths of Angstroms. We hope that our studies will help direct researchers pursuing more applied goal. In the long term, these studies and their follow-ups we believe will be important in guiding an ability to design new enzymes to carry out reactions that are beneficial to society.
