Determination of Enzymatic Activity

To examine the activity of the intra-molecular CoA transfer of Mct, a spectrophotometric assay was used. Mesaconyl-C4-CoA has a higher extinction coefficient at 290 nm (ε_290 nm = 5800 M^–1 cm^–1) than mesaconyl-C1-CoA (ε_290 nm = 2900 M^–1 cm^–1). Therefore, the conversion of mesaconyl-C1-CoA was measured by the increase in absorbance at 290 nm (Δε_290 nm = 2900 M^–1 cm^–1). To conduct the measurements, 150 μL assay volume (200 mM HEPES/KOH, pH_25 °C 8.0, 22 nM Mct, and varying concentrations of mesaconyl-CoA) was incubated at 55 °C, and change in absorbance at 290 nm was monitored over time in a 3 mm quartz cuvette. The reaction was started with the substrate (ranging from 50 to 1600 μM for mesaconyl-C1-CoA and 40 to 1300 μM for mesaconyl-C4-CoA).

To test for alternative CoA acceptors, Mct was preincubated in reaction buffer (200 mM HEPES, pH 7.5) and supplemented with 20 mM of the corresponding carboxylic acid. After 5 min of preincubation, the reaction was started by the addition of 1 mM mesaconyl-C1-CoA. Samples were taken after 0 and 20 min and stopped on ice by the addition of HCl to a final concentration of 100 mM. The precipitated enzyme was removed by centrifugation (4 °C and 17 000g), and the supernatants were analyzed by HPLC-MS and for the presence of alternative CoA thioesters.

To evaluate and quantify the kinetics of succinate as acceptor acids, an enzyme assay was performed (55 μL; 200 mM HEPES/KOH, pH_25 °C 8.0, 6 μM Mct, 1 mM mesaconyl-C4-CoA, and varying concentrations of succinate). The reaction was started with the addition of succinate and incubated for 20 min at 55 °C. At 0, 1, and 20 min, a sample of 5 μL was taken and quenched in 45 μL of formic acid. The precipitated enzyme was removed by centrifugation (4 °C and 17 000g), and the supernatants were analyzed by HPLC-MS for the presence of succinyl-CoA.

Determination of CoA thioesters was performed using a HiRes-LC-MS. The chromatographic separation was performed on a Thermo Scientific Vanquish HPLC system using a Kinetex Evo C18 column (150 × 2.1 mm², 100 A, 1.7 μm, Phenomenex) equipped with a 20 × 2.1 mm² guard column of similar specificity at a constant eluent flow rate of 0.25 mL/min and a column temperature of 25 °C with eluent A being 50 mM ammonium formate at a pH of 8.1 water and eluent B being MeOH (Honeywell). The injection volume was 1 μL. The elution profile consisted of the following steps and linear gradients: 0–2 min constant at 0% B; 2–10 min from 0 to 80% B; 10–12 min constant at 80% B; 12–12.1 min from 80 to 0% B; and 12.1–15 min constant at 0% B. A Thermo Scientific ID-X Orbitrap mass spectrometer was used in positive mode with an electrospray ionization source and the following conditions: ESI spray voltage 3500 V, sheath gas at 50 arbitrary units, auxiliary gas at 10 arbitrary units, sweep gas at 1 arbitrary unit, ion transfer tube temperature at 300 °C, and vaporizer temperature at 350 °C. Detection was performed in full-scan mode using the orbitrap mass analyzer at a mass resolution of 240 000 in the mass range 800–900 (m/z). Extracted ion chromatograms of the [M + H]⁺ forms were integrated using Tracefinder software (Thermo Scientific). Absolute concentrations for succinyl-CoA were calculated based on an external calibration curve.