Shoun H, Beppu T, Arima K
J Biol Chem. 1980 Oct 10;255(19):9319-24.
p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) was rapidly inactivated by treatment with phenylglyoxal, by a process obeying pseudo-first order kinetics. The reaction with the reagent was also examined by amino acid analyses, radioactivity measurements, and spectrophotometric analyses. Results of these analyses were consistent with each other, which shows that the inactivation was due to modification of argiine residue(s). Addition of saturating amounts of p-hydroxybenzoate (or benzoate) during the treatment resulted in marked protection of the enzyme from the inactivation as well as a significant decrease in modification of arginine residues, while phenol showed no effect. Modification in the absence of p-hydroxybenzoate caused a spectral change in the flavin moiety of the enzyme similar to that due to the enzyme.substrate complex formation, and losses in both the overall activity and the substrate-binding ability accompanied the spectral change. On the other hand, such spectral change was not observed and the substrate-binding ability was retained even after the overall activity had decreased to a great extent when p-hydroxybenzoate as added during the modification treatment. These results suggest that phenylglyoxal (an analogue of the substrate) was incorporatd into the substrate-binding site and that an arginine residue is involved in the site, having an interaction with the carboxylate anion of the substrate.
对羟基苯甲酸羟化酶（EC 1.14.13.2）经苯乙二醛处理后会迅速失活，该反应遵循准一级动力学。还通过氨基酸分析、放射性测量和分光光度分析对该酶与试剂的反应进行了研究。这些分析结果相互一致，表明失活是由于精氨酸残基的修饰。在处理过程中添加饱和量的对羟基苯甲酸（或苯甲酸）可显著保护酶不被失活，同时精氨酸残基的修饰也显著减少，而苯酚则无此作用。在无对羟基苯甲酸的情况下进行修饰会导致酶的黄素部分发生光谱变化，类似于因酶 - 底物复合物形成而产生的光谱变化，并且总体活性和底物结合能力的丧失都伴随着这种光谱变化。另一方面，当在修饰处理过程中添加对羟基苯甲酸时，即使总体活性大幅下降，也未观察到这种光谱变化，并且底物结合能力得以保留。这些结果表明苯乙二醛（底物类似物）被纳入底物结合位点，并且一个精氨酸残基参与该位点，与底物的羧酸根阴离子相互作用。
