Isothermal Titration Calorimetry Uncovers Substrate Promiscuity of Bicupin Oxalate Oxidase

<p>Oxalate oxidase from <em>Ceriporiopsis subvermispora</em> (CsOxOx) is a manganese-dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to form two moles of carbon dioxide and one mole of hydrogen peroxide. CsOxOx is the first oxalate oxidase reported to have a two beta barrel architecture (bicupin). The CsOxOx catalyzed oxidation of oxalate reaction can be monitored by coupling the product H₂O₂ to the horseradish peroxidase-catalyzed oxidation of 2,2'-azinobis-(3- ethylbenzthiazoline-6-sulphonic acid) and by membrane inlet mass spectrometry. Coupled enzyme assays often confound data interpretation. Isothermal titration calorimetry (ITC) measures heat changes that occur during a reaction. Therefore, ITC can measure reaction rates where both the substrates and products are spectrophotometrically silent and when the reaction products are unknown. In this work, we apply the multiple injection method of ITC to characterize the catalytic properties of oxalate oxidase. Steady-state kinetic constants using oxalate as the substrate determined by multiple injection ITC are comparable to those obtained by the horseradish peroxidase coupled assay and by membrane inlet mass spectrometry. Furthermore, we used this technique to identify mesoxalate as a substrate for the CsOxOx-catalyzed reaction, with kinetic parameters comparable to that of oxalate, and to identify a number of small molecule carboxylic acid compounds that also serve as substrates for the enzyme.</p>

Name thesis:degree_name

Master of Science in Chemical Sciences (MSCB)

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Chemistry

Year dc:date.available

2016

Author dc:creator

• Rana, Hassan Ali

Contributors dc:contributor

• Ellen W. Moomaw
• Daniela Tapu
• Michael Van Dyke

• oxalate oxidase
• enzyme kinetics
• isothermal titration calorimetry
• Chemistry

Repository record dc:identifier

https://digitalcommons.kennesaw.edu/mscs_etd/7

OAI identifier oai:identifier

oai:digitalcommons.kennesaw.edu:mscs_etd-1006