Functional Dynamics during Induced-fit Enzyme Turnover

DESCRIPTION (provided by applicant): Motions are key to the action of many proteins. Dynamics are poorly understood, especially, in the micro- second and slower regimes that often limit turnover. They lie beyond most experimental and computational methods. Our long term objective is a fundamental understanding of how conformational dynamics underpin mechanism. This will be achieved by characterizing structurally and kinetically the motions of individual amino acids and their relation to function in the turnover cycle of an induced-fit enzyme. Recently developed methods of NMR relaxation dispersion analysis will be applied to a model enzyme to determine conformational exchange rates. These will be integrated with high resolution crystal structures and NMR residual dipolar coupling characterization of faster dynamics. Arginine kinase presents examples of domain and loop movements that are substrate-induced, as well as inherent concerted motions. Both the NMR spectra and x-ray diffraction of arginine kinase are of unusually high quality for a representative enzyme of this size (42 kDa). This presents a unique opportunity to elucidate the structure-dynamics- function correlates of motions representative of those that are functionally important for many proteins. The emphasis will be on mapping the backbone conformational exchange rates for each residue, particularly in the micro-/milli-second regime. NMR relaxation exchange rates will be measured during enzyme turnover at various equilibrium substrate/product concentrations to determine which motions are associated with binding, dissociation or reaction steps. Crystal structures will be determined for the stable enzyme complexes within the random sequential bi-bi mechanism, and NMR relaxation titration will be used to determine the exchange rates for conformational changes associated with binding or dissociation. Thus, the structural changes and time constants of motion will be dissected for each amino acid through the steps of the turnover cycle. The functional role of the motions will be established through joint analysis of kinetics and dynamics following temperature, mutational and other perturbations of the enzyme. Health Relevance: A fundamental understanding of molecular motion is essential to understanding how human proteins achieve their normal function and the mechanisms through which disease may result from genetic mutations.