Intermolecular and Intramolecular Stable Isotope Studies in Alanine

Citation

Dallas, Brooke Hillary (2024) Intermolecular and Intramolecular Stable Isotope Studies in Alanine. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/xqdx-pp86. https://resolver.caltech.edu/CaltechTHESIS:05252024-201300283

Abstract

In Chapter 1 of this thesis, we give an introduction to this body of work, providing some background for context.

In Chapter 2, we present a set of theoretical predictions for the carbon isotope distribution between equilibrated carbon sites of alanine and pyruvate. We start with the simplest possible theoretical treatment, and work progressively through higher levels of theory, showing consistency in the direction and magnitude of expected fractionation across these treatments.

In Chapter 3, we present our experimental work to confirm the predictions made in Chapter 2 by measuring the δ¹³C of the α carbon site in alanine that has undergone equilibration with the analogous carbon site in pyruvate via the alanine transaminase enzyme (ALT).

In Chapter 4, we describe the process that led to our (re)discovery of β-hydrogen-deuterium exchange in amino acids catalyzed by transaminases. We then provide a literature review on the small body of historical work on this system, which took place primarily during the 1960s and 70s. This literature summary provides the background necessary for the reader to appreciate our experimental work presented in the next chapter.

In Chapter 5, we present novel ¹H NMR and ¹³C NMR experimental observations of intermolecular hydrogen isotope exchange between water and the α and β carbon sites of alanine, as well as intramolecular hydrogen isotope exchange between the α and β carbons, all of which is catalyzed by alanine transaminase (ALT). These experiments track the abundances of eight isotopically distinct alanine species varying in their position and/or number of hydrogen isotopes over a series of reactions differing in initial alanine isotopic composition and initial water isotopic composition. With the data collected we are able to determine up to thirteen rate constants and ten equilibrium constants describing the transfer of hydrogen and deuterium amongst these eight isotopic variants and water, as well as the thermodynamic equilibrium constants between them.

Thesis Files