Friebolin H, Baumann W, Hauck M, Kurz D, Wajda R, Weisshaar G, Keilich G, Ziegler D, Brossmer L, von Nicolai H
Hoppe Seylers Z Physiol Chem. 1984 Nov;365(11):1309-21.
The specificities of one viral and five bacterial sialidases were investigated by 1H-NMR-spectroscopy with substrates or substrate mixtures containing two sialic acid residues of different linkage types. This technique allows - in contrast to the methods used before - the simultaneous determination of the rates of hydrolysis of both NeuAc linkages in a single experiment. The substrate specificities of the enzymes are discussed on the basis of the relation of the rate constants k/k'. The data obtained are more exact and more informative than those of separate experiments as reported previously. Among the enzymes investigated, i.e. sialidases of fowl plague virus (FPV = VKH), Clostridium perfringens (CP), Vibrio cholerae (VC), Bifidobacterium bifidum var. pennsylvanicum (BBif), Bifidobacterium lactentis (BLac), and Arthrobacter ureafaciens (AU), the activity of the viral sialidase VKH shows the highest, the activities of the Bifidobacterium sialidases the lowest dependence on the nature and on the linkage type of the different substrates. All sialidases preferentially cleave the NeuAc alpha 2-3-Gal linkage with the exception of the enzyme of Arthrobacter ureafaciens (AU) which shows a higher affinity to alpha 2-6 linkages. However, this does not apply to the side-arm-linked NeuAc alpha 2-6 structure in NeuAc alpha 2-3 Gal beta 1-3 (NeuAc alpha 2-6)-GlcNAc beta 1-3Gal beta 1-4Glc (Substrate B). This substrate in generally cleaved very slowly and is hardly affected by the viral enzyme. After the alpha 2-3 linkage, the alpha 2-8 bond in NeuAc alpha 2-8 NeuAc alpha 2-3 Gal beta 1-4Glc(Substrate A) is most susceptible for the sialidases VKH, CP and VC. An elongation of the carbohydrate chain (Substrate D) is accompanied by a reduction of the rate of cleavage for all enzymes. The experiments with alpha 1-acid glycoprotein, fetuin, and with the glycopeptides obtained by proteolytic degradation of the latter, revealed the same specificity towards the alpha 2-3 and the alpha 2-6 linkages as the oligosaccharides. Influenced by the chemical nature and the size of the substrate, NeuAc is released from the native alpha 1-acid glycoprotein more quickly than from the corresponding glycopeptide. All sialidases investigated so far are strictly exo-enzymes as could be demonstrated by the cleavage of NeuAc alpha 2-8 NeuAc alpha 2-3 Gal beta 1-4Glc (Substrate A).
利用含不同连接类型的两个唾液酸残基的底物或底物混合物，通过1H - NMR光谱研究了一种病毒唾液酸酶和五种细菌唾液酸酶的特异性。与之前使用的方法相比，该技术能够在单个实验中同时测定两种NeuAc连接的水解速率。基于速率常数k/k'的关系讨论了这些酶的底物特异性。所获得的数据比之前报道的单独实验的数据更精确、更具信息量。在所研究的酶中，即禽瘟病毒（FPV = VKH）、产气荚膜梭菌（CP）、霍乱弧菌（VC）、宾夕法尼亚双歧杆菌变种（BBif）、乳酸双歧杆菌（BLac）和脲节杆菌（AU）的唾液酸酶，病毒唾液酸酶VKH的活性最高，双歧杆菌唾液酸酶的活性对不同底物的性质和连接类型的依赖性最低。除脲节杆菌（AU）的酶对α2 - 6连接具有更高亲和力外，所有唾液酸酶均优先切割NeuAcα2 - 3 - Gal连接。然而，这不适用于NeuAcα2 - 3 Galβ1 - 3（NeuAcα2 - 6）- GlcNAcβ1 - 3Galβ1 - 4Glc（底物B）中侧链连接的NeuAcα2 - 6结构。该底物通常切割非常缓慢，且几乎不受病毒酶的影响。在α2 - 3连接之后，NeuAcα2 - 8 NeuAcα2 - 3 Galβ1 - 4Glc（底物A）中的α2 - 8键对唾液酸酶VKH、CP和VC最敏感。碳水化合物链的延长（底物D）伴随着所有酶切割速率的降低。用α1酸性糖蛋白、胎球蛋白以及通过对后者进行蛋白水解降解得到的糖肽进行的实验，揭示了与寡糖对α2 - 3和α2 - 6连接相同的特异性。受底物化学性质和大小的影响，NeuAc从天然α1酸性糖蛋白中释放的速度比从相应糖肽中更快。如通过切割NeuAcα2 - 8 NeuAcα2 - 3 Galβ1 - 4Glc（底物A）所证明的，迄今为止研究的所有唾液酸酶都是严格的外切酶。
