Acyl-CoA Thioesterase Activity Assay.

Lipids dissolved in ethanol were incubated with purified M. musculus ΔNterm-Them1 or thioesterase domains and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) or 30 min at 37 °C in assay buffer containing 30 mM Hepes pH 7.5, 150 mM NaCl, and 5% glycerol. Myristoyl-CoA was added to a protein–DTNB–lipid mixture to initiate reaction in a total reaction volume of 200 μL/well, with final protein concentration at 1 μM, DTNB at 300 μM, lipid at 25 μM or 50 μM, and myristoyl-CoA ranging from 0 to 20 μM. Absorbance readings at 412 nm were read every 10 s for 1 h. The initial velocities were plotted against substrate concentrations and fitted with the Michaelis–Menten equation to yield the maximum velocity (V_max) and K_m (the Michaelis constant) using GraphPad Prism 8.0. Each experiment was conducted with two technical replicates for each sample and repeated three times.

Full methods are provided in SI Appendix, SI Materials and Methods.