Estrada P, Mata I, Dominguez J M, Castillón M P, Acebal C
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, Spain.
Biochim Biophys Acta. 1990 Mar 26;1033(3):298-304. doi: 10.1016/0304-4165(90)90137-l.
beta-Glucosidase is a key enzyme in the hydrolysis of cellulose to D-glucose. beta-Glucosidase was purified from cultures of Trichoderma reesei QM 9414 grown on wheat straw as carbon source. The enzyme hydrolyzed cellobiose and aryl beta-glucosides. The double-reciprocal plots of initial velocity vs. substrate concentration showed substrate inhibition with cellobiose and salicin. However, when p-nitrophenyl beta-D-glucopyranoside was the substrate no inhibition was observed. The corresponding kinetic parameters were: K = 1.09 +/- 0.2 mM and V = 2.09 +/- 0.52 mumol.min-1.mg-1 for salicin; K = 1.22 +/- 0.3 mM and V = 1.14 +/- 0.21 mumol.min-1.mg-1 for cellobiose; K = 0.19 +/- 0.02 mM and V = 29.67 +/- 3.25 mumol.min-1.mg-1 for p-nitrophenyl beta-D-glucopyranoside. Studies of inhibition by products and by alternative product supported an Ordered Uni Bi mechanism for the reaction catalyzed by beta-glucosidase on p-nitrophenyl beta-D-glucopyranoside as substrate. Alternative substrates as salicin and cellobiose, a substrate analog such as maltose and a product analog such as fructose were competitive inhibitors in the p-nitrophenyl beta-D-glucopyranoside hydrolysis.
β-葡萄糖苷酶是将纤维素水解为D-葡萄糖的关键酶。β-葡萄糖苷酶是从以麦草为碳源培养的里氏木霉QM 9414中纯化得到的。该酶可水解纤维二糖和芳基β-葡萄糖苷。初始速度与底物浓度的双倒数图表明，纤维二糖和水杨苷存在底物抑制作用。然而，当以对硝基苯基β-D-吡喃葡萄糖苷为底物时，未观察到抑制作用。相应的动力学参数为：水杨苷的Km = 1.09±0.2 mM，Vmax = 2.09±0.52 μmol·min⁻¹·mg⁻¹；纤维二糖的Km = 1.22±0.3 mM，Vmax = 1.14±0.21 μmol·min⁻¹·mg⁻¹；对硝基苯基β-D-吡喃葡萄糖苷的Km = 0.19±0.02 mM，Vmax = 29.67±3.25 μmol·min⁻¹·mg⁻¹。产物抑制和替代产物抑制的研究支持了β-葡萄糖苷酶以对硝基苯基β-D-吡喃葡萄糖苷为底物催化反应的有序单双机制。水杨苷和纤维二糖等替代底物、麦芽糖等底物类似物以及果糖等产物类似物在对硝基苯基β-D-吡喃葡萄糖苷水解中均为竞争性抑制剂。
