DYNAMICS IN ENZYME ACTION

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This Program Project proposal concerns the elucidation of the role of polypeptide chain dynamics in the mechanism in the mechanism of catalysis by enzymes. The overall goal of the proposal is to explore the hypothesis that polypeptide chain dynamics strongly influence the local interactions that mediate enzyme action, and may be an essential factor that allows to achieve high turnover rates as well as exquisite specificity in their reactions. The Program Project is divided into 4 projects, all tightly focused on this long-term goal, and closely tied to each other. Two very different enzyme systems, dihydrofolate reductase (DHFR) and a metallo- beta-lactamase, may have been chosen for intensive study, in the hope that results obtained for the two systems may be generalizable in terms of the guiding hypothesis of the proposal. The first two projects are concerned with experimental measures to determine polypeptide chain dynamics using NMR relaxation and other methods. Project 1 (P.E. Wright, P.I.) will focus on the dynamics using NMR relaxation and other methods. Project 1 (P.E. Wright, P.I.) will focus on the dynamics of DHFR in binary and ternary complexes with substrates, inhibitors and cofactors, and on mutants specifically designed on the basis of preliminary NMR dynamics measurements. A similar protocol will ultimately be followed in Project 2 (H,J. Dyson, P.I.) for the metallo-beta-lactamase. Initial goals for this project will involve resonance assignment and correlation with structure, together with estimates of the dynamics from 15N and 13C relaxation and from amide proton hydrogen exchange rates. Project 3 (S.J. Benkovic, P.I.) will utilize the information provided in Projects 1 and 2 to design site- directed mutants and inhibitors for the two enzymes. This information besides being vital for the enzymes are important drug targets, DHFR for anti-cancer therapy and the metallo-beta-lactamase because it is a major component of antibiotic resistance in pathogenic bacteria. Project 4 (D.A. Case, P.1., C.L. Brooks, III, Co-P.I.) provides the essential theoretical underpinning for the project. Based on information available from Projects 1, 2 and 3, Project 4 will attempt to model reaction pathways consistent with structural, dynamic and kinetic information, providing the potential for a fundamental understanding of reaction mechanisms at an atomic and electronic level. Together, these projects constitute a focused effort to understand enzyme catalysis, incorporating the novel idea that dynamics of the polypeptide may well be an integral and important component of the entire process.