The Adaptive Nature of Cytochrome P4502E1-Mediated Ethanol Oxidation: Implications on the Overall Catalytic Scheme

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Katherine Grace Ryan (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Advisor
Gregory Raner

Abstract: The Cytochrome P450 family is a class of enzymes responsible for the breakdown of about 75% of the drugs that are administered (18). The 2E1 isoform of this family is known as the "ethanol-inducible" member, because ethanol has demonstrated the capacity to affect the metabolism of other 2E1 substrates (15, 16). It has been proposed that elevated concentrations of ethanol in the liver are responsible for the activation of acetaminophen and nitrosamine constituents found in cigarette smoke into hepatotoxic byproducts, and that an increase in polarity in a very hydrophobic active site may have a significant role in perpetuating these toxic effects (2-3, 23). In the present study, the objective was to probe the influence of ethanol and subsequently, increased active site polarity, on the catalytic scheme of other P4502E1 substrates. Additionally, due to the fact that many other P450 isoforms contain hydrophobic active sites and therefore, may also be affected by changing polarity in the active site environment, the implications of these studies were evaluated in the context of the P450 family as a whole. Potential evidence regarding the existence of an effector site adjacent to the catalytic site, and its role in 2E1 catalysis, was also probed. Designed to examine the effect of ethanol on the previously observed substrate inhibition pattern in p-nitrophenol oxidation, results from current studies indicated that ethanol potentially disrupts ligand-ligand interactions between the catalytic and effector sites. The ability of ethanol to potentially alter rate-limiting steps in 2E1-mediated benzyl alcohol oxidation was explored through intrinsic isotope effect studies. Despite being limited by the purity of the benzyl alcohol substrate, an apparent shift or "unmasking" of the isotope effect was observed when ethanol was added. Finally, reversibility studies were carried out to evaluate the capacity of ethanol to interfere with 2E1 inactivation by 4-nitrobenzaldehyde, a potent irreversible inhibitor of the 2B4 isoform (7). When ethanol was present in low concentrations, a synergistic effect was exhibited, where the activity of 2E1 was reduced to an even greater degree than reactions containing the aldehyde alone. An increase in enzyme activity was observed in reactions containing the aldehyde and higher concentrations of ethanol, relative to those containing only the aldehyde, suggesting that ethanol exerted a pseudo-protective effective in this case.

Additional Information

Publication
Thesis
Language: English
Date: 2010
Keywords
Chemistry, Biochemistry
Subjects
Cytochrome P-450.
Ethanol.
Oxidation.
Enzymes.
Catalysis.