Enzymes direct chemical reactions with precision and speed, making life as we know it possible. How they do this is still not completely understood, but the relatively recent discovery of subpicosecond protein motion coupled to the reaction coordinate has provided a crucial piece of the puzzle. This type of motion is called a rate-promoting vibration (RPV) and has been seen in a number of different enzymatic systems. It typically involves a compression of the active site of the enzyme which lowers the barrier for the reaction to occur. In this work we present a number of studies that probe these motions in two dehydrogenase enzymes, yeast alcohol dehydrogenase (YADH) and homologs of lactate dehydrogenase (LDH). The goal of the study on the reaction of YADH was to probe the role of the protein in proton tunneling in the enzyme, which was suggested to occur from experimental kinetic isotope effect studies. We did this using transition path sampling (TPS), which perturbatively generates ensembles of reactive trajectories to observe transitions between stable states, such as chemical reactions. By applying a quantum method that can account for proton tunneling, centroid molecular dynamics, and generating reactive trajectory ensembles with and without the method, we were able to observe the change in barrier to proton transfer upon application of the tunneling method. We found that there was little change in the barrier, showing that classical over-the-barrier transfer is dominant over tunneling in the proton transfer in YADH. We also applied the knowledge of RPVs to identify a new class of allosteric molecules, which modulate enzymatic reaction not by changing a binding affinity, but by disrupting the reactive motion of enzymes. We showed, through design of a novel allosteric effector for human heart LDH, applying TPS to a system with and without the small molecule bound, and analysis of the reaction coordinate of the reactive trajectory ensemble, that the molecule was able to disrupt the motion of the protein such that it was no longer coupled to the reaction. We also examined the subpicosecond motions of two other LDHs, from Plasmodium falciparum and Cryptosporidium parvum, which evolved separately from previously studied LDHs. We found, using TPS and reaction coordinate identification, that while the LDH from C. parvum had similar dynamics to the earlier LDHs, the LDH from P. falciparum had a earlier transition-state associated with proton transfer, not hydride transfer. This is likely due to this LDH having a larger active site pocket, increasing the amount of motion necessary for proton transfer, and, thus, the barrier to proton transfer. More work is necessary in this system to determine whether the protein is coupled with the search for the reactive conformation for proton transfer. Protein motion coupled to the particle transfer in dehydrogenases plays an important role in their reactions and there is still much work to be done to understand the extent and role of RPVs.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/620868 |
| Date | January 2016 |
| Creators | Dzierlenga, Michael W., Dzierlenga, Michael W. |
| Contributors | Schwartz, Steven D., Bandarian, Vahe, Lichtenberger, Dennis L., Sanov, Andrei, Schwartz, Steven D. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Electronic Dissertation |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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