ABSTRACT

Title: Distal Residues in Enzyme Catalysis and Protein Design

Enzyme engineering -the capability to design proteins to act as catalysts for particular, desired chemical reactions- is currently in its very early stages. This project seeks to develop design principles for enzyme engineering, using properties of the protein structure that can be computed. This project builds on very recent discoveries that reveal new information about how nature builds the catalytic center of a protein molecule, wherein multiple layers of amino acids within the protein structure provide the necessary properties that enable chemical reactions to happen within living organisms at physiological temperature and under mild conditions. Many of these same reactions, when performed in a laboratory or industrial setting, require high temperature and caustic conditions. An important, ultimate goal of this work is to be able to design protein catalysts to perform industrial chemical reactions, because for most industrial chemical processes, there is no natural enzyme that can serve as a catalyst. The development of such protein catalysts for industrial use will translate to less energy usage, lower costs, less waste, and fewer unwanted by-products. Thus the ability to design protein catalysts has many potential benefits to the environment, to the economy, and to human well-being. This project will train doctoral students and undergraduate research interns, including members of underrepresented groups, to become highly qualified scientists in the areas of biochemistry and computational biology; the cultivation of such expertise is vital to the regional high-tech economy and to U.S. competitiveness in the global economy.


This project will explore how distal residues contribute to enzyme catalysis, establish additional principles about their role in catalysis, and take the first steps toward using these principles for enzyme design. The project takes a multilateral approach, combining theory, computation, biochemical experiments, x-ray crystal structure determination, x-ray solution scattering, and high-field electron spin resonance spectroscopy. These simulations and experiments will provide information about the electrostatic, structural, and dynamic effects of amino acid residues, including remote residues, on catalysis. The specific examples to be studied in this project, a Y-family DNA polymerase DinB and an aldolase, were chosen because they lead into - and provide insight into - protein design problems. The effects of distal residues on proton transfer equilibria in the active site, and the associated requirements for catalysis, will be investigated. Study of the roles of individual residues in Y-family DNA polymerases will increase understanding of the mechanism of extension in DNA replication and repair of damaged DNA. The results will be used to address whether improved extension capability can be engineered into the polymerase DinB. Investigation of the interactions between residues in a natural aldolase will increase understanding of its catalytic mechanism; the emerging principles will be used to improve activity of an artificially designed retroaldolase. New features to address the problem of enzyme design, namely the predictability and importance of distal residue participation in forging the right catalytic properties and the use of coupled protonation states, are introduced. The capability to design enzymes that can catalyze any desired chemical reaction is a grand challenge in science. This project will develop design principles to build on the knowledge base that is necessary to create such enzymes. Enzyme design principles to be developed and tested in this project thus have potential impact on biotechnology, environmental remediation, agriculture, and the growth of a "green" economy, as well as the chemical industry.

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