Date of Award
Spring 3-23-2017
Track
Biochemistry
Degree Type
Thesis
Degree Name
Master of Science in Chemical Sciences (MSCB)
Department
Chemistry
Committee Chair/First Advisor
Dr. Ellen Moomaw
Committee Member
Dr. Michael Van Dyke
Committee Member
Dr. Christopher Dockery
Abstract
Oxalate oxidase is a manganese containing enzyme that catalyzes the oxygen dependent oxidation of oxalate to carbon dioxide and hydrogen peroxide. Oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx) is the first characterized bicupin oxalate oxidase. Potential applications of oxalate oxidase in cancer treatment, paper pulping, and biofuel cells highlight the need to study the effects of hydrogen peroxide on the CsOxOx catalyzed oxidation of oxalate. Membrane inlet mass spectrometry (MIMS) was used to directly measure initial rates of carbon dioxide formation and oxygen consumption in varying initial concentrations of hydrogen peroxide. This work demonstrates that hydrogen peroxide is both a reversible noncompetitive inhibitor of the CsOxOx catalyzed oxidation of oxalate and an irreversible inactivator. CsOxOx is irreversibly inactivated under turnover conditions and hydrogen peroxide plays a key role in this turnover dependent inactivation. The introduction of catalase to reaction mixtures protects the enzyme from inactivation allowing reactions to proceed to completion. Circular dichroism spectra suggest that hydrogen peroxide does not induce changes in CsOxOx global protein structure. Additionally, we show that the CsOxOx mediated mesoxalate reaction consumes oxygen and produces hydrogen peroxide, which is in contrast with previous proposals that the enzyme catalyzed the non-oxidative decarboxylation of this three carbon substrate. Finally, we observe a 13Ckinetic isotope effect on the CsOxOx catalyzed reaction using MIMS which supports that the carbon-carbon bond cleavage of oxalate is at least partially rate limiting.