Gómez M, Isorna P, Rojo M, Estrada P
Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain.
Biochimie. 2001 Oct;83(10):961-7. doi: 10.1016/s0300-9084(01)01341-4.
The variation of kinetic parameters of beta-xylosidase from Trichoderma reesei QM 9414 with pH was used to elucidate the chemical mechanism of the p-nitrophenyl beta-D-xylopyranoside hydrolysis. The pH-dependence of V and V/K(m) showed that a group on the enzyme with a pK value of 3.20 must be unprotonated and a group with a pK value of 5.20 must be protonated for activity and both are involved in catalysis. Solvent-perturbation studies indicated that these groups are neutral acid type. Temperature dependence of kinetic parameters suggested the stickiness of the substrate at lower temperatures than the optimum and the calculated ionization enthalpies pointed to carboxyl groups as responsible for both pKs. Chemical modification with triethyloxonium tetrafluoroborate and protection with the substrate studies demonstrated essential carboxyl groups on the enzyme. Profiles of pK(i) for D-gluconic acid lactone indicated that a group with a pK value of 3.45 must be protonated for binding and it has been assigned to the carboxyl group of D-gluconic acid formed by lactone ring breakdown in solution.
利用里氏木霉QM 9414的β-木糖苷酶动力学参数随pH的变化来阐明对硝基苯基β-D-吡喃木糖苷水解的化学机制。V和V/K(m)对pH的依赖性表明，酶上一个pK值为3.20的基团必须去质子化，一个pK值为5.20的基团必须质子化才能具有活性，且二者均参与催化作用。溶剂扰动研究表明这些基团为中性酸类型。动力学参数的温度依赖性表明，在低于最适温度时底物具有粘性，计算得到的电离焓表明羧基是两个pK值的原因。用四氟硼酸三乙氧鎓进行化学修饰以及用底物进行保护研究证明了酶上存在必需的羧基。D-葡萄糖酸内酯的pK(i)曲线表明，一个pK值为3.45的基团必须质子化才能结合，并且它已被指定为由溶液中内酯环分解形成的D-葡萄糖酸的羧基。
