1H-NMR-Spektroskopie. Spezifität mikrobieller Sialidasen gegenüber komplexen Substraten

Publisher Website
Google Scholar
Abstract
The specificities of 1 viral and 5 bacterial sialidases were investigated by 1H-NMR-spectroscopy with substrates or substrate mixtures containing 2 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 [N-acetyl-neuraminic acid] 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''. Among the enzymes investigated, i.e., sialidases of fowl plague virus (FPV = VKH), Clostridium perfringens (CP), Vibrio cholera (VC), Bifidobacterium bifidum var. pennsylvaniculm (BBif), Bifidobacterium lactentis (BLAC), and Arthrobacter ureafaciens (AU), the activity of the viral sialidase VKH shows the highest, the activities of Bifidobacterium sialidases the lowest dependence of 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 AU which shows a higher affinty to .alpha.2-6 linkages. However, this does not apply to the side-arm-linked NeuAc.alpha.2-6 structure in NeuAc.alpha.2-3Gal.beta.1-3(NeuAc.alpha.2-6)-GlcNAc.beta.1-3Gal.beta.1-4Glc (Substrate B). This substrate is 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.2-8NeuAc.alpha.2-3Gal.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 exoenzymes as could be demonstrated by the cleavage of NewAc.alpha.2-8NeuAc.alpha.2-3Gal.beta.1-4Glc (Substrate A).