A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme–substrate complex.

中文翻译：

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工业β-葡萄糖苷酶的片段衍生调节剂

使用动力学测定法对560个市售片段的文库进行的片段筛选确定了一个小分子，该小分子增加了真菌糖苷水解酶TrBgl2的活性。通过目录法进行的类似物和详细的动力学分析确定了改进的化合物，其充当非必需激活剂，最大激活量最多增加2倍。该化合物没有激活相关的细菌糖苷水解酶CcBglA，显示出特异性。有趣的是，初始片段的类似物显然通过混合模型机制抑制了TrBgl2和CcBglA。尽管无法确定激活剂与55 kDa TrBgl2结合的晶体结构，但溶液NMR实验证明了该激活剂的特异性结合位点。NMR光谱的部分分配给出了该位点氨基酸的身份，从而建立了TrBgl2激活模型。激活剂在底物结合位点的入口处结合，为酶-底物复合物产生有效的构象。