title: Membrane inlet mass spectrometry reveals that Ceriporiopsis subvermispora bicupin oxalate oxidase is inhibited by nitric oxide.
abstract: Membrane inlet mass spectrometry (MIMS) uses a semipermeable membrane as an inlet to a mass spectrometer for the measurement of the concentration of small uncharged molecules in solution. We report the use of MIMS to characterize the catalytic properties of oxalate oxidase (E.C. 1.2.3.4) from Ceriporiopsis subvermispora (CsOxOx). Oxalate oxidase is a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The MIMS method of measuring OxOx activity involves continuous, real-time direct detection of oxygen consumption and carbon dioxide production from the ion currents of their respective mass peaks. (13)C2-oxalate was used to allow for accurate detection of (13)CO2 (m/z 45) despite the presence of adventitious (12)CO2. Steady-state kinetic constants determined by MIMS are comparable to those obtained by a continuous spectrophotometric assay in which H2O2 production is coupled to the horseradish peroxidase catalyzed oxidation of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid). Furthermore, we used MIMS to determine that NO inhibits the activity of the CsOxOx with a KI of 0.58±0.06 μM.
authors: Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, GA 30144, USA. Electronic address: emoomaw@kennesaw.edu.
authors: Ellen W
authors: EW
authors: Moomaw
authors: Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, GA 30144, USA.
authors: Richard
authors: R
authors: Uberto
authors: Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USA.
authors: Chingkuang
authors: C
authors: Tu
mesh_terms: D002384:Catalysis; D055453:Coriolaceae / Q000201:enzymology*; D004789:Enzyme Activation; D004867:Equipment Design; D017022:Flow Injection Analysis / Q000295:instrumentation*; D013058:Mass Spectrometry / Q000295:instrumentation*; D008567:Membranes, Artificial; D009569:Nitric Oxide / Q000737:chemistry*; D019815:Oxalic Acid / Q000737:chemistry*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D013048:Specimen Handling / Q000295:instrumentation
chemical_list: D008567:Membranes, Artificial; D009569:Nitric Oxide; D019815:Oxalic Acid; D010088:Oxidoreductases; C020598:oxalate oxidase
keywords: Cupin; Enzyme assay; Membrane inlet mass spectrometry; NO inhibition; Oxalate oxidase; Pichia pastoris