| For all the scientific knowledge that has been gained in the field of enzymology over the last century, there are still many aspects about enzymes' catalytic ability remain intriguing. Commonly achieving significant rate enhancements over their uncatalyzed reaction enzymes have become a scientific curiosity due to their catalytic achievements. By exploiting mechanistic facilitators such as electrostatic charge interactions, quantum mechanical tunneling, and the use of general acids and bases and metals, enzymes have a plethora of tools available to lower the energy of its necessary bio-reaction. One such area of interest is examining the prevalence and extent quantum mechanical hydrogen tunneling plays in enzyme mechanisms. Of the hundreds of hydrogen transfer reactions that enzymes catalyze, how often is tunneling utilized as a possible mechanistic pathway to lower the energy barrier? In order to examine this, as well as other mechanistic features, three different enzymes that catalyze hydrogen transfer reactions were studied. These medically relevant enzymes, glutamate dehydrogenase and a mesophilic and thermophilic dihydrofolate reductase offer stark mechanistic differences and intriguing comparisons between the chemistry catalyzed and the catalyst.;To reveal mechanistic details, kinetic isotope effects (KIEs) were employed. Using this kinetic isotope effect data it is possible to analyze and determine the extent of hydrogen tunneling described by various models. Acquisition of KIE data was performed by a mixed labeling experiment, using protium (H), deuterium (D), and tritium (T) labeling on the ubiquitous cofactor, NADPH. Synthesis of this labeled cofactor and its long term preservation was a significant task and is explored in great detail. In addition to KIE and hydrogen tunneling data, enzyme kinetic aspects such as intrinsic KIEs and commitments to catalysis can be calculated. From this research, a great deal of comprehension about a few enzyme catalyzed, hydrogen transfer reactions can be gained which will be beneficial to experimentalists and theoreticians. |
