Meinke A, Damude H G, Tomme P, Kwan E, Kilburn D G, Miller R C, Warren R A, Gilkes N R
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
J Biol Chem. 1995 Mar 3;270(9):4383-6. doi: 10.1074/jbc.270.9.4383.
In the commonly accepted mechanism for enzymatic hydrolysis of cellulose, endo-beta-1,4-glucanases randomly cleave glucosidic bonds within glucan polymers, providing sites for attack by exo-cellobiohydrolases (EC 3.2.1.91). It has been proposed that hydrolysis by Trichoderma reesei cellobiohydrolase II is restricted to the ends of cellulose polymers because two surface loops cover its active site to form a tunnel. In a closely related endoglucanase, E2 from Thermomonospora fusca, access to the substrate appears to be relatively unhindered because the carboxyl-proximal loop is shortened, and the amino-proximal loop is displaced. The hypothesis was examined by deletion of a region in Cellulomonas fimi cellobiohydrolase A corresponding to part of the carboxyl-proximal loop of T. reesei cellobiohydrolase II. The mutation enhanced the endoglucanase activity of the enzyme on soluble O-(carboxymethyl)cellulose and altered its activities on 2',4'-dinitrophenyl-beta-D-cellobioside, insoluble cellulose, and cellotetraose.
在普遍接受的纤维素酶促水解机制中，内切-β-1,4-葡聚糖酶随机切割葡聚糖聚合物中的糖苷键，为外切纤维二糖水解酶（EC 3.2.1.91）提供攻击位点。有人提出，里氏木霉纤维二糖水解酶II的水解作用仅限于纤维素聚合物的末端，因为两个表面环覆盖其活性位点形成一个通道。在一种密切相关的内切葡聚糖酶，即来自嗜热单孢菌的E2中，由于羧基近端环缩短且氨基近端环移位，底物的进入似乎相对不受阻碍。通过缺失纤维单胞菌纤维二糖水解酶A中与里氏木霉纤维二糖水解酶II羧基近端环部分相对应的区域来检验这一假设。该突变增强了该酶对可溶性O-（羧甲基）纤维素的内切葡聚糖酶活性，并改变了其对2',4'-二硝基苯基-β-D-纤维二糖苷、不溶性纤维素和纤维四糖的活性。
