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

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ABSTRACT Enzymes have important implications for understanding many human diseases as well as for developing new medicines and therapies. Design of small molecule drugs without side-effects targeting enzymes and designer enzymes as biotherapeutics are widely pursued in the pharmaceutical industry. However, these endeavors are hindered, among other aspects, by the lack of fundamental understanding of enzyme function including the factors that enable enzymes to achieve high catalytic efficiencies. For more than a century, an immense wealth of information has been accumulated based on experimental and computational investigations. Collectively, the biochemical model of enzyme catalysis has revealed the vital roles of active-site residues and other secondary structure elements. However, clear understanding of the roles of: (1) the functionally important conformational sub-states (or rare intermediates); (2) the distal regions including the conserved residues and surface loops; and (3) the surrounding solvent, in enzyme catalysis still remain elusive. For close to two decades, Agarwal lab has been working on using joint computational-experimental approaches for obtaining answers to several important questions about enzymes. Investigations of >20 different enzyme systems have enabled us to contribute to building a biophysical model of enzyme catalysis, which is improving our knowledge of these highly efficient molecular machines. We have discovered conserved network of residues linking surface loop regions to the active-site in several medically important enzyme systems, and successfully developed and validated quasi-anharmonic analysis (QAA) method for identification of conformational sub- states. In this proposal, we describe computational investigations of several enzymes including human ribonucleases, dihydrofolate reductase and biliverdin reductase. Using previously developed and new approaches, the following key questions will be answered: (1) What roles do conformational sub-states play in enzyme catalysis? Specifically, functionally important higher energy sub-states and their linkage to kinetics of the rate-limiting step in enzyme cycle will be quantitatively characterized; (2) Energy flow within preferential pathways (or network channels) formed by conserved residues will be characterized as the biophysical mechanism for long-distance coupling; (3) Thermodynamical coupling between the surrounding environment (solvent) and the enzyme structure and catalyzed reaction will be characterized. A combination of molecular dynamics (MD) and new theoretical analysis methods will be used. We have and continue to work with a number of experimental laboratories to validate our models and their outputs. Experimental data from NMR, enzyme kinetics, X-ray and other techniques on wild type and mutant versions of enzyme systems will be used to iteratively refine our models. These investigations will provide new insights into mechanism of long-distance effects and insights into factors that contribute to the catalytic efficiency of enzymes. The developed software will continue to be made available to the community and we will support a wide variety of labs in their investigations of enzymes. Over long-term these efforts will lead to designing of better allosteric modulators and designer enzymes for biotherapies.