The long-term objectives of this research are to determine the roles of protein structure, flexibility, adaptability and dynamics in enzyme catalysis. The overall hypothesis is that residues at or distant from the active site contribute to catalysis by affecting protein motions and fluidity, conformational changes, and subunit interactions. Horse liver alcohol dehydrogenase is a good system for studying structural and dynamics effects on catalysis. Three-dimensional structures of various complexes have been determined by X-ray crystallography for two conformational states, and rate and dissociation constants for each step in the mechanism, including the chemical step (hydride transfer), can be estimated from steady-state and transient kinetics. Site-directed mutagenesis will be used to substitute amino acid residues in different regions of the enzyme, and the effects on the catalytic mechanism, structure and dynamics will be quantitatively evaluated. (1) Residues in the hinge region between the catalytic and coenzyme binding domains and the domain contact regions will be altered in order to change the rate and extent of the conformational change, and allosteric interactions through the subunits will be studied with heterodimeric enzymes. (2) Amino acid residues that may contribute to protein promoting vibrations and residues buried in core regions will be mutated in order to change the protein fluidity and dynamics. """"""""Extraneous"""""""" structural elements or Q-loops will be deleted to test the role of the protein scaffold. (3) Three-dimensional structures of wild-type and mutated enzymes complexed with substrate analogs will be determined by X-ray crystallography at high resolution, and dynamic information will be extracted by analysis of translation, libration, screw-rotation displacements (TLS parameters) for domains and subdomains that cooperate in catalysis. The dynamics hypothesis will be supported if the directions and amplitudes of motion are correlated with rates of hydride transfer. The kinetic, structural and dynamic results should improve our understanding of catalysis and facilitate rational drug design. Redesign of enzymes for medical and industrial applications should become more """"""""rational"""""""" when the connections between protein structure and catalysis are better understood. Development of new catalysts based on protein scaffolds will benefit from a better understanding of protein motions and dynamics. Furthermore, the design of therapeutic agents must incorporate information about protein motions, as specificities are affected by amino acid residues that are distant from the active site. Studies on substrate and inhibitor specificities of alcohol dehydrogenases suggest that active sites are adaptable and that binding affinities are not easily explained with a static structure. Design of better inhibitors for treating the pathology of alcoholism depends on understanding the dynamics of catalysis by the rate-limiting enzyme in the pathway of alcohol metabolism. ? ? ?
