Nagainis M P, Pu W, Cheng B, Taylor K E, Schmidt D E
Biochim Biophys Acta. 1981 Jan 15;657(1):203-11. doi: 10.1016/0005-2744(81)90144-3.
The pH-dependence of fumarylacetoacetase (4-fumarylacetoacetate fumaryl-hydrolase, EC 3.7.1.2) activity was studied in the pH range 6.25-8.50. After correction of the substrate concentration for enolate formation, the Michealis constant was found to be pH independent in this range. Likewise, the Ki values for the competitive inhibitors chloride and fluoride were found to be independent of pH between 6.25-8.50. A bell-shaped curve described the log V vs. pH dependence, and ionization constants of 6.5 and 8.2 were calculated. Tentatively an imidazole group and a sulfhydryl group were assigned to the constants 6.5 and 8.2, respectively. Both p-hydroxymercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid) react with both sulfhydryl groups per subunit in the native protein and three sulfhydryl groups per subunit in the denatured protein. Substrate protects one sulfhydryl group in the native protein from reaction with 5,5'-dithiobis(2-nitrobenzoid acid). Substrate or the competitive inhibitor, fluoride, protect the enzyme from inactivation by p-hydroxymercuribenzoate. In addition p-hydroxymercuribenzoate shows saturation kinetics. Neither sulfhydryl inhibitor completely inactivates the enzyme. The enzyme is described as having three sulfhydryl groups per subunit, one of which is inaccessible to the sulfhydryl specific reagents when the protein is in the native state. One of the two accessible sulfhydryl groups is either near the active site or essential in maintaining the structure of the protein.
在pH范围6.25 - 8.50内研究了富马酰乙酰乙酸酶（4 - 富马酰乙酰乙酸富马酰水解酶，EC 3.7.1.2）的活性与pH的关系。在对烯醇化物形成的底物浓度进行校正后，发现米氏常数在此范围内与pH无关。同样，竞争性抑制剂氯离子和氟离子的Ki值在6.25 - 8.50之间也与pH无关。一条钟形曲线描述了log V与pH的关系，并计算出电离常数为6.5和8.2。初步将咪唑基团和巯基分别指定给常数6.5和8.2。对羟基汞苯甲酸和5,5'-二硫代双（2 - 硝基苯甲酸）都能与天然蛋白质中每个亚基的两个巯基以及变性蛋白质中每个亚基的三个巯基发生反应。底物可保护天然蛋白质中的一个巯基不与5,5'-二硫代双（2 - 硝基苯甲酸）反应。底物或竞争性抑制剂氟化物可保护酶不被对羟基汞苯甲酸灭活。此外，对羟基汞苯甲酸呈现饱和动力学。两种巯基抑制剂都不能使酶完全失活。该酶被描述为每个亚基有三个巯基，当蛋白质处于天然状态时，其中一个巯基对巯基特异性试剂不可接近。两个可接近的巯基中的一个要么靠近活性位点，要么对维持蛋白质结构至关重要。
