间羟苯甲酸4-羟化酶的化学计量学研究

Stoichiometry of meta Hydroxybenzoate 4 Hydroxylase

用扫描分光光度计对酶所催化的反应进行化学计量学研究。在反应开始后的不同时间，在２００～４００ｎｍ波长范围内扫描，观察到不同的吸收波谱，直到吸收波谱不再改变为止，由此得到间羟苯甲酸４羟化酶催化间羟苯甲酸转变成３，４二羟苯甲酸随时间变化的吸收波谱。用摩尔消光系数计算每种成分的浓度，在２次独立的实验中，底物与产物的摩尔比值分别为１：０．９９８和１：１．０８５，提示来源于Ｃｏ．ｔｅｓｔｏｓｔｅｒｏｎｉＫＨ１２２３Ｓ菌株的ＭＯＢ４ＨＯａｓｅ与文献报道的来源于Ａｓｐｅｒｇｉｌｕｓｎｉｇｅｒ菌株的ＭＯＢ４ＨＯａｓｅ所催化的反应是相同的。

Stoichiometry of catalytic reaction by enzyme can be researched by scanning spectrophotometry. The difference absorption spectra at wave lengths between 200 and 400 nm were observed after starting the reaction at different time until no further change in the absorption profile could be detected.Therefore it was obtained that the absorption spectra of conversion of m hydroxybenzoate to protocatechuate by meta hydroxybenzoate 4 hydroxylase changed following the time. Concentration of each component was calculated from the molar extinction coefficient. The molar ratioes of substrate to product in two independent experiments were 1:0.998 and 1:1.085 respectively,indicating a good accord to the reaction by the MOB4 HOase extracting from Co. testosteroni KH122 3S and Aspergillus niger genera.