Biophysical Model of Enzyme Catalysis: Conformational sub-states, solvent coupling and energy networks

PI: David Ussery, Oklahoma State University

Project Summary

Enzymes have important implications for understanding many human diseases and for developing new medicines and therapies. The design of enzyme-targeting small-molecule drugs with minimal side effects, as well as designer enzymes as biotherapeutics, is widely pursued in the pharmaceutical industry. However, these efforts are hindered, in part, by a limited fundamental understanding of enzyme function, including the factors that enable enzymes to achieve high catalytic efficiencies.

For more than a century, extensive knowledge has been accumulated through experimental and computational investigations. Collectively, the biochemical model of enzyme catalysis has revealed the vital roles of active-site residues and secondary structural elements. However, a clear understanding of the roles of: (1) functionally important conformational substates or rare intermediates; (2) distal regions, including conserved residues and surface loops; and (3) the surrounding solvent in enzyme catalysis remains elusive. Our group has used integrated computational and experimental approaches to address important questions about enzyme function. Investigations of more than 20 different enzyme systems have enabled us to contribute to the development of a biophysical model of enzyme catalysis, improving our understanding of these highly efficient molecular machines. We have discovered conserved residue networks linking surface-loop regions to active sites in several medically important enzyme systems, and we have developed and validated the quasi-anharmonic analysis method for identifying conformational substates. In this proposal, we describe computational investigations of several enzymes, including human ribonucleases, dihydrofolate reductase, and biliverdin reductase. Using previously developed and new approaches, we will address the following key questions: (1) What roles do conformational substates play in enzyme catalysis? Specifically, functionally important high-energy substates and their links to the kinetics of rate-limiting steps in the enzyme cycle will be quantitatively characterized. (2) How does energy flow through preferential pathways or network channels formed by conserved residues, and how does this contribute to long-distance coupling? (3) How does thermodynamic coupling between the surrounding solvent, enzyme structure, and catalyzed reaction influence enzyme function?A combination of molecular dynamics simulations and new theoretical analysis methods will be used. We have worked, and will continue to work, with several experimental laboratories to validate our models and their predictions. Experimental data from NMR, enzyme kinetics, X-ray crystallography, and other techniques using wild-type and mutant enzyme systems will be used to iteratively refine our models.These investigations will provide new insights into the mechanisms of long-distance effects in enzymes and the factors that contribute to catalytic efficiency. The software developed through this work will continue to be made available to the community, and we will support a wide range of laboratories in their enzyme investigations. Over the long term, these efforts will contribute to the design of improved allosteric modulators and designer enzymes for biotherapeutic applications.