Processivity of Cellobiohydrolases Is Limited by the Substrate
Processivity of Cellobiohydrolases Is Limited by the Substrate
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DOI：
10.1074/jbc.m110.161059
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发表时间：
2011-01-07
影响因子：
4.8
通讯作者：
Vaeljamaee, Priit
中科院分区：
文献类型：
--
作者：
Kurasin, Mihhail;Vaeljamaee, Priit
过程纤维素生物水解酶（CBHs）是真菌纤维素酶系统的关键组成部分。尽管有丰富的结构数据证实了作用的过程模式，但关于CBHs的过程性的定量信息很少。在这里，我们开发了一种测量纤维素酶加工率的方法。用二氨基吡啶标记后，对纤维素上酶生成的不溶性还原基团进行灵敏的荧光检测，可以定量还原端外模式和内模式引发的数量。来自里氏木霉的CBHs TrCel7A和来自黄孢平革菌的PcCel7D均采用了外模式和内模式的还原端并行起始。测定的TrCel7A和PcCel7D对纤维素水解的处理能力值比催化常数（k(cat)）和解离速率常数（k(off)）的比值得到的固有处理能力值低一个数量级以上。我们提出，纤维素链上的无障碍路径的长度限制了CBHs对纤维素的加工能力。TrCel7A和PcCel7D的k（off）值不同，而k（cat）值相似。此外，内切葡聚糖酶（EGs）的k（off）值远高于CBHs的k（off）值，而EGs和CBHs的k（cat）值在同一数量级内。这些结果表明，k（off）的值可能是选择纤维素酶的主要目标。
Processive cellobiohydrolases (CBHs) are the key components of fungal cellulase systems. Despite the wealth of structural data confirming the processive mode of action, little quantitative information on the processivity of CBHs is available. Here, we developed a method for measuring cellulase processivity. Sensitive fluorescence detection of enzyme-generated insoluble reducing groups on cellulose after labeling with diaminopyridine enabled quantification of the number of reducing-end exo-mode and endo-mode initiations. Both CBHs TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium employed reducing-end exo- and endo-mode initiation in parallel. Processivity values measured for TrCel7A and PcCel7D on cellulose hydrolysis were more than an order of magnitude lower than the values of intrinsic processivity that were found from the ratio of catalytic constant (k(cat)) and dissociation rate constant (k(off)). We propose that the length of the obstacle-free path available for a processive run on cellulose chain limits the processivity of CBHs on cellulose. TrCel7A and PcCel7D differed in their k(off) values, whereas the k(cat) values were similar. Furthermore, the k(off) values for endoglucanases (EGs) were much higher than the k(off) values for CBHs, whereas the k(cat) values for EGs and CBHs were within the same order of magnitude. These results suggest that the value of k(off) may be the primary target for the selection of cellulases.