Natural sialoside analogues for the determination of enzymatic rate constants
Natural sialoside analogues for the determination of enzymatic rate constants
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DOI：
10.1039/b613909d
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发表时间：
2006-01-01
影响因子：
3.2
通讯作者：
Bennet, Andrew J.
中科院分区：
文献类型：
--
作者：
Indurugalla, Deepani;Watson, Jacqueline N.;Bennet, Andrew J.
合成了两种异构体 4-甲基伞形基-α-D-N-乙酰神经氨酰吡喃半乳糖苷（1 和 2）。这些化合物含有天然的 α-2,3 或 α-2,6 唾液酸-半乳糖基键，以及连接的 4-甲基伞形酮，以方便检测其水解。这些化合物被设计为天然唾液酸苷类似物，用于唾液酸酶活性的连续测定，其中唾液酸酶催化的反应与释放的半乳糖苷的外切-β-半乳糖苷酶催化的水解相结合，产生游离的4-甲基伞形酮。使用来自绿色小单孢菌的野生型和亲核突变体 Y370G 重组唾液酸酶测量 1 和 2 的动力学参数。使用新的连续测定法测量的这些类似物的动力学参数与天然底物 3'-唾液酸乳糖的参数非常一致。鉴于选择了具有多种最佳 pH 值的市售外切-β-半乳糖苷酶，这种新方法用于表征野生型唾液酸酶与天然唾液酸苷类似物 1 的完整 pH 曲线。因此，在连续测定模式中使用这些新底物 1 和 2（可通过 UV/Vis 或荧光光谱检测），使得对具有天然唾液酸苷键的唾液酸酶活性的表征变得更加容易。
Two isomeric 4-methylumbelliferyl-alpha-D-N-acetylneuraminylgalactopyranosides ( 1 and 2) were synthesised. These compounds contain either the natural alpha-2,3 or alpha-2,6 sialyl - galactosyl linkages, as well as an attached 4-methylumbelliferone for convenient detection of their hydrolyses. These compounds were designed as natural sialoside analogues to be used in a continuous assay of sialidase activity, where the sialidase-catalysed reaction is coupled with an exo-beta-galactosidase-catalysed hydrolysis of the released galactoside to give free 4-methylumbelliferone. The kinetic parameters for 1 and 2 were measured using the wild-type and nucleophilic mutant Y370G recombinant sialidase from Micromonospora viridifaciens. Kinetic parameters for these analogues measured using the new continuous assay were in good agreement with the parameters for the natural substrate, 3'-sialyl lactose. Given the selection of commercially available exo-beta-galactosidases that possess a variety of pH optima, this new method was used to characterise the full pH profile of the wild-type sialidase with the natural sialoside analogue 1. Thus, use of these new substrates 1 and 2 in a continuous assay mode, which can be detected by UV/Vis or fluorescence spectroscopy, makes characterisation of sialidase activity with natural sialoside linkages much more facile.