Enzymatic activity assay for purified HMGCS2

The spectrophotometric method described by Clinkenbeard et al (23) was used with modifications. Each protein sample (16.4-43.2 µg) was incubated in 900 µl enzyme assay buffer (100 mM Tris-HCl, 100 µM EDTA, 0.2% v/v Triton X-100, pH 8.2) containing 300 µM acetyl-CoA (Sigma-Aldrich; Merck KGaA) for 17 min at 30˚C to prevent inactivation of HMGCS2 by succinylation (24,25). Next, 35 nmol acetoacetyl-CoA (Sigma-Aldrich; Merck KGaA) were added, where HMGCS2 activity was calculated from the rate of decrease of acetoacetyl-CoA measured by spectrophotometry at 300 nm, at which the absorbance of the enolate form of acetoacetyl-CoA was maximum (26). The molar extinction coefficient of acetoacetyl-CoA is 3.6x10³ in this enzyme assay buffer (23). Activity measurements were performed in three independent experiments. Data are expressed as the mean ± SD.