van der Bolt F J, Drijfhout M C, Eppink M H, Hagen W R, van Berkel W J
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Protein Eng. 1994 Jun;7(6):801-4. doi: 10.1093/protein/7.6.801.
p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains five sulfhydryl groups per subunit. Cysteine-->serine replacements show that the thiols are not essential for catalysis. The increased dissociation constant for FAD in mutant Cys158Ser suggests that Cys158 is important for the solvation of the pyrophosphate moiety of the prosthetic group. Wild-type p-hydroxybenzoate hydroxylase is rapidly inactivated by mercurial compounds. Inactivation by a spin-labeled derivative of p-chloromercuribenzoate is fully abolished in mutant Cys211Ser. Incorporation of the spin label in the other Cys-->Ser mutants strongly impairs substrate binding without affecting the catalytic properties of the FAD. The results are discussed with respect to previous tentative assignments from chemical modification studies and in light of the 3-D structure of the enzyme-substrate complex.
来自荧光假单胞菌的对羟基苯甲酸羟化酶每个亚基含有五个巯基。半胱氨酸向丝氨酸的替换表明，这些硫醇对于催化并非必不可少。突变体Cys158Ser中FAD的解离常数增加，这表明Cys158对于辅基焦磷酸部分的溶剂化很重要。野生型对羟基苯甲酸羟化酶会被汞化合物迅速灭活。在突变体Cys211Ser中，对氯汞苯甲酸的自旋标记衍生物的灭活作用完全消除。在其他Cys→Ser突变体中掺入自旋标记会严重损害底物结合，而不会影响FAD的催化特性。根据先前化学修饰研究的初步结果以及酶-底物复合物的三维结构，对结果进行了讨论。
