Isothermal titration calorimetry uncovers substrate promiscuity of bicupin oxalate oxidase from Ceriporiopsis subvermispora

Abstract

Isothermal titration calorimetry (ITC) may be used to determine the kinetic parameters of enzyme-catalyzed reactions when neither products nor reactants are spectrophotometrically visible and when the reaction products are unknown. We report here the use of the multiple injection method of ITC to characterize the catalytic properties of oxalate oxidase (OxOx) from Ceriporiopsis subvermispora (CsOxOx), a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The multiple injection ITC method of measuring OxOx activity involves continuous, real-time detection of the amount of heat generated (dQ) during catalysis, which is equal to the number of moles of product produced times the enthalpy of the reaction (ΔH_app). Steady-state kinetic constants using oxalate as the substrate determined by multiple injection ITC 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-sulfonic acid) and by membrane inlet mass spectrometry. Additionally, we used multiple injection ITC to identify mesoxalate as a substrate for the CsOxOx-catalyzed reaction, with a 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.

Keywords: ABTS, 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid).; CsOxOx, OxOx from Ceriporiopsis subvermispora; Cupin; Enzyme kinetics; HRP, horseradish peroxidase; Isothermal titration calorimetry; OxOx, oxalate oxidase; Oxalate oxidase.

Fig. 1

Heat flow as a function of time for the CsOxOx-catalyzed complete conversion of oxalate into products gives the ΔH_app of reaction. After equilibration at 25 °C (not shown), the reaction was initiated by the addition of 1 µL of 2 mM potassium oxalate, pH 4.0 into 1 µM CsOxOx (180 µL) in 50 mM sodium succinate, pH 4.0 (first arrow). This injection was repeated at 500 s (second arrow).

Fig. 2

Thermogram monitoring the power required to maintain isothermal conditions of 20 successive injections of 2 µL of 20 mM oxalate into 112 nM oxalate oxidase (180 µL) previously thermally equilibrated at 25 °C. Under these conditions, when the enzyme velocity reaches a new steady state, the power remains constant. The inset corresponds to the first four injections of substrate into the sample cell and the arrows represent the decrease in the heat flow (dQ/dt) required to maintain thermal equilibrium at each steady state condition.

Fig. 3

Rate versus oxalate concentration data from Fig. 2 fitted to the Michaelis–Menten equation to give kinetic parameters provided in Table 1. The inset shows the same data in a double reciprocal plot yielding the nearly identical values of k_cat and K_m as the fit to the Michaelis–Menten equation.

Fig. 4

The heat flow as a function of time for the reaction catalyzed by CsOxOx using mesoxalate as substrate measured in conditions of steady-state kinetics at 25 °C. The sample cell contained 180 µL of 456 nM CsOxOx and 20 injections of 2.5 µL of 10 mM mesoxalate were performed with an interval of 180 s between them. The inset shows the rate versus mesoxalate concentration data fitted to the Michaelis–Menten equation to give kinetic parameters provided in Table 2 for mesoxalate.