| An understanding of the molecular basis for enzyme catalysis is the objective of molecular enzymology. Enzyme catalysis involves transformation of substrate and recycling of enzyme. The contributions of this dissertation address both of these criterions. Catalytic antibodies attest to the appropriateness of transition-state theory for enzyme catalysis. To overcome low efficiencies observed for catalytic antibodies, combinatorial libraries offer the potential to create larger pools of potential catalysts. As a test of this approach, a library was constructed by combining the 43C9 heavy chain with a light chain library derived from the immune response to a phosphoramidate hapten. Surprisingly, the light chains of four library antibodies demonstrated high sequence homology ({dollar}>{dollar}92%) to 43C9. These 43C9-like antibodies possessed similar binding and catalytic properties as 43C9. These variants of the 43C9 catalytic anti body provided insight into the utility of the rigid antibody scaffold and transition-state complementarity to hydrolyze a substrate. Unlike catalytic antibodies, Escherichia coli dihydrofolate reductase (DHFR) displays significant flexibility. The role of DHFR flexibility in catalysis was investigated applying two approaches. Since the composition of loop regions defines their respective properties, site-directed mutagenesis was employed to alter the flexible properties of four regions, the {dollar}alpha{dollar}E-{dollar}beta{dollar}E, Met20, {dollar}beta{dollar}C-{dollar}beta{dollar}D, and {dollar}beta{dollar}F-{dollar}beta{dollar}G loops. Mutants of the hinge {dollar}alpha{dollar}E-{dollar}beta{dollar}E loop, suggest that domain rotation is dominated by a shear mechanism. Mutation at the base of the Met20 loop only affects the formation of binary complexes, not ternary complexes occurring in the catalytic cycle. Based solely on observed dynamical properties, alterations of the {dollar}beta{dollar}C-{dollar}beta{dollar}D loop did not produce significant effects on binding and the rate of hydride transfer, suggesting that the dynamical properties of this loop are incidental. The binding and catalytic properties of {dollar}beta{dollar}F-{dollar}beta{dollar}G loop mutants confirmed the importance of interloop interactions modulating Met20 loop conformations. As an alternative approach to studying a role for protein flexibility, catalysis by DHFR was examined under steady-state and pre-steady-state conditions as a function of urea. These results support prior data indicating urea activation involves a localized denaturation. A three-state model is proposed that predicts the nature of the data. |
