Activity assay of O-Acetyl-L-homoserine sulfhydrylase (CaMet15p).

The study aimed to obtain conditions optimal for the activity of O-acetyl-L-homoserine sulfhydrylase (CaMet15p) and a method suitable for its measurement. The selection of appropriate reaction conditions included the identification of the reaction buffer and its pH, substrate and enzyme concentrations. The activity of CaMet15p was measured by the detection of product L-homocysteine (HCT) via LC/MS system with pre-column derivatization. Mass spectra were recorded using an Agilent 6470A triple quadrupole (6470 Triple Quad LC/MS, Agilent Technologies, Waldbronn, Germany) LC/MS system (1260 Infinity II, Agilent Technologies,Waldbronn, Germany) with electrospray ionization source (ESI). HCT reacts with 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) to produce a HCT-TNB complex, that can be also detected by the diode array detector (HPLC-DAD). Optimal conditions for HCT-TNB complex detection included identification of mobile phase, elution profile, injection volume, and most suitable wavelengths for the detection have been identified. This dataset contains the description of the methodology, and the set of selected chromatographs obtained for control as well as enzymatic reaction samples.The studies were financially supported by the project OPUS 20 financed by the National Science Centre (No. UMO-2020/39/B/NZ7/01519).