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).

本研究旨在获取O-乙酰-L-高丝氨酸巯基酶（O-acetyl-L-homoserine sulfhydrylase，CaMet15p）活性的最优反应条件，并建立适配其活性检测的方法。反应条件的筛选涵盖了反应缓冲液及其pH值、底物与酶浓度的确定。CaMet15p的活性通过柱前衍生化法结合液相色谱-质谱（LC/MS）系统检测产物L-高半胱氨酸（L-homocysteine，HCT）得以测定。质谱数据通过配备电喷雾电离源（electrospray ionization source，ESI）的安捷伦6470A三重四极杆液相色谱-质谱联用系统（6470 Triple Quad LC/MS，安捷伦科技公司，德国瓦尔德布龙）及1260 Infinity II液相色谱系统（安捷伦科技公司，德国瓦尔德布龙）采集。L-高半胱氨酸可与5,5'-二硫代双(2-硝基苯甲酸)（5,5’-dithiobis(2-nitrobenzoic acid)，DTNB）反应生成HCT-TNB复合物，该产物亦可通过高效液相色谱-二极管阵列检测器（high performance liquid chromatography-diode array detector，HPLC-DAD）进行检测。针对HCT-TNB复合物检测的最优条件涵盖了流动相、洗脱程序、进样体积的筛选，同时确定了适配的检测波长。本数据集包含本研究方法的详细说明，以及针对对照组与酶促反应样本所获取的筛选后色谱图集合。本研究获得了波兰国家科学中心资助的OPUS 20项目（项目编号：UMO-2020/39/B/NZ7/01519）的经费支持。