Murata Takeomi, Hattori Takeshi, Amarume Satoshi, Koichi Akiko, Usui Taichi
Department of Applied Biological Chemistry, Shizuoka University, Japan.
Eur J Biochem. 2003 Sep;270(18):3709-19. doi: 10.1046/j.1432-1033.2003.03757.x.
Novel chromogenic substrates for endo-beta-galactosidase were designed on the basis of the structural features of keratan sulfate. Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (2), which consists of two repeating units of N-acetyllactosamine, was synthesized enzymatically by consecutive additions of GlcNAc and Gal residues to p-nitrophenyl beta-N-acetyllactosaminide. In a similar manner, GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (1), GlcNAcbeta1-3Galbeta1-4Glcbeta-pNP (3), Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta-pNP (4), Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glcbeta-pNP (5), and Galbeta1-6GlcNAcbeta1-3Galbeta1-4Glcbeta-pNP (6) were synthesized as analogues of 2. Endo-beta-galactosidases released GlcNAcbeta-pNP or Glcbeta-pNP in an endo-manner from each substrate. A colorimetric assay for endo-beta-galactosidase was developed using the synthetic substrates on the basis of the determination of p-nitrophenol liberated from GlcNAcbeta-pNP or Glcbeta-pNP formed by the enzyme through a coupled reaction involving beta-N-acetylhexosaminidase (beta-NAHase) or beta-d-glucosidase. Kinetic analysis by this method showed that the value of Vmax/Km of 2 for Escherichia freundii endo-beta-galactosidase was 1.7-times higher than that for keratan sulfate, indicating that 2 is very suitable as a sensitive substrate for analytical use in an endo-beta-galactosidase assay. Compound 1 still acts as a fairly good substrate despite the absence of a Gal group in the terminal position. In addition, the hydrolytic action of the enzyme toward 2 was shown to be remarkably promoted compared to that of 4 by the presence of a 2-acetamide group adjacent to the p-nitrophenyl group. This was the same in the case of a comparison of 1 and 3. Furthermore, the enzyme also catalysed a transglycosylation on 1 and converted it into GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (9) and GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (10) as the major products, which have N-acetyllactosamine repeating units.
基于硫酸角质素的结构特征设计了新型的内切β-半乳糖苷酶显色底物。由对硝基苯基β-N-乙酰乳糖胺连续添加GlcNAc和Gal残基酶促合成了由两个N-乙酰乳糖胺重复单元组成的Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-pNP（2）。以类似方式，合成了GlcNAcβ1-3Galβ1-4GlcNAcβ-pNP（1）、GlcNAcβ1-3Galβ1-4Glcβ-pNP（3）、Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ-pNP（4）、Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ-pNP（5）和Galβ1-6GlcNAcβ1-3Galβ1-4Glcβ-pNP（6）作为2的类似物。内切β-半乳糖苷酶以内切方式从每个底物释放出GlcNAcβ-pNP或Glcβ-pNP。基于对通过β-N-乙酰己糖胺酶（β-NAHase）或β-d-葡萄糖苷酶的偶联反应由该酶形成的GlcNAcβ-pNP或Glcβ-pNP释放的对硝基苯酚的测定，使用合成底物开发了一种用于内切β-半乳糖苷酶的比色测定法。通过该方法的动力学分析表明，弗氏埃希氏菌内切β-半乳糖苷酶对2的Vmax/Km值比对硫酸角质素的高1.7倍，表明2非常适合作为内切β-半乳糖苷酶测定分析用的灵敏底物。尽管化合物1在末端位置没有Gal基团，但它仍然是相当好的底物。此外，与4相比，由于在对硝基苯基相邻位置存在2-乙酰酰胺基团显著促进了该酶对2的水解作用。1和3的比较情况也是如此。此外，该酶还催化1上的转糖基化反应，并将其转化为主要产物GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-pNP（9）和GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-pNP（10），它们具有N-乙酰乳糖胺重复单元。
