Date of Degree
PhD (Doctor of Philosophy)
This thesis describes an effort to expand current knowledge of catalysis in biological systems. The focus is on understanding how enzymes activate covalent bonds and specifically to study C-H bond activation via enzymes. The work presented here examined the role of protein dynamics and hydrogen tunneling in enzyme catalysis. Dihydrofolate reductase from Escherichia coli (ecDHFR), which catalyzes a single hydride transfer reaction, was selected as the model system for these studies. Intrinsic kinetic isotope effects (KIEs) have been shown to be highly sensitive probes in examining the chemical steps of enzymatic reactions, especially since they can indirectly infer the role of certain dynamic fluctuations in the ecDHFR-catalyzed reaction. This study provides evidence in support of the phenomenological Marcus-like model presently well accepted amongst both experimental enzymologists and some members of the computational community — this model suggests that certain molecular fluctuations prevail during the enzyme-catalyzed hydrogen transfer reaction and assist the chemical step. Previous studies of the temperature-dependence of KIEs focused on examining the network of residues that are dynamically linked to the hydride-transfer step and are localized far from the active site; a network initially proposed by computational studies. This thesis, on the other hand, focuses on the effect of the active site environment on the C-H→C transfer. While no spectroscopy experiments were performed to measure the dynamics in the active site, sensitive kinetic experiments were used to examine the physical features of the C-H→C transfer via rigorous perturbation of the donor-acceptor-distance (DAD). KIE measurements on a series of carefully designed active site mutants have been interpreted using a Marcus-like model and complemented by results obtained via molecular dynamic simulations and X-ray crystallography. Active site mutants were designed to alter the DAD and its dynamics in a controlled manner with a minimal effect on the active-site electrostatics. The results suggest that the mutations have affected the reorganization energy necessary for the system to reach the transition state and have modulated the average DAD as well as its distribution at the transition state. The study on the active-site mutants was extended on N23PP—a dynamically altered mutant that was the source of an extensive debate in the field due to opposing views regarding the altered dynamics and its role in assisting the hydride transfer step. Findings presented in this thesis indicate that temperature dependent kinetic complexity masked the intrinsic KIEs in the earlier studies, and that our methodology revealed the significant differences between the natures of the hydride transfer catalyzed by the WT and by the dynamically impaired mutant. Collectively these results further our understanding of the role of enzyme dynamics and quantum tunneling in enhancing enzymatic reactions. In the future these results will be correlated to findings from vibrational spectroscopy, high-level calculations and NMR studies (as they become available) in order to establish the structure-dynamic-function-relationship both in ecDHFR and in enzymes in general.
xvi, 176 pages
Includes bibliographical references (pages 160-176).
Copyright 2012 Vanja Stojkovic