| This thesis presents preliminary work done in an effort to link enzyme kinetics and dynamics on femtosecond-picosecond timescale. Formate dehydrogenase, which catalyzes hydride transfer reaction, is selected as the model system for these studies. Kinetic isotope effects and three-pulse echo peak shift spectroscopy were the techniques employed to study the kinetics and dynamics respectively. The work done provides evidence in support of the Marcus-like model, which is presently well accepted amongst enzymologists. These studies provide information about the molecular fluctuations that prevail during an enzyme catalyzed hydrogen transfer reaction. The photon echo results for the ternary complexes show a lack of static contribution in the frequency-frequency correlation function and the intrinsic kinetic isotope effects exhibit no temperature dependence. These results suggest that, once the enzyme is near the transition state for the hydride transfer step, the structure around the active site is very well organized with the precise arrangement of the active site residues involved in the atom transfer. This optimized structure imposes severe constrains on the active site, making it rigid (motions on nanosecond time scale or slower, <0.03 cm-1, are not sensed by the chromophore). As a result, near the transition state only low amplitude, fast fluctuations are observed that sample all the conformations within a few picoseconds and no slow dynamics are observed. The photon echo results of the binary complex suggest that moving away from the transition state relaxes the constrains on the active site structure, and slower motions that are required to bring the system to the transition state become apparent. This novel approach of studying the kinetics and dynamics simultaneously corroborates the hypothesis made by the Marcus-like model and lays ground for future work, where dynamically altered mutants can be tested using this approach. |
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