Kinetic studies on endo-β-galactosidase by a novel colorimetric assay and synthesis of N-acetyllactosamine-repeating oligosaccharide β-glycosides using its transglycosylation activity

被引:12
作者
Murata, T [1 ]
Hattori, T [1 ]
Amarume, S [1 ]
Koichi, A [1 ]
Usui, T [1 ]
机构
[1] Shizuoka Univ, Dept Appl Biol Chem, Shizuoka 4228529, Japan
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2003年 / 270卷 / 18期
关键词
endo-beta-galactosidase; enzyme assay; kinetics; poly (N-acetyllactosamine); transglycosylation; HAMSTER-KIDNEY CELLS; ESCHERICHIA-FREUNDII; ENZYMATIC-SYNTHESIS; BACTEROIDES-FRAGILIS; HELICOBACTER-PYLORI; KERATAN SULFATE; FLAVOBACTERIUM-KERATOLYTICUS; CARBOHYDRATE STRUCTURE; ENDOGLYCOSIDIC ACTION; BACILLUS-CIRCULANS;
D O I
10.1046/j.1432-1033.2003.03757.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-acetyl-lactosamine, 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), GlcNA cbeta1-3Galbeta1-4Glcbeta-pNP (3), Gal beta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta-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 GlcNAcbbeta-NP 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 V-max/K-m 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 GlcNAc beta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (9) and GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (10) as the major products, which have N-acetyllactosamine repeating units.
引用
收藏
页码:3709 / 3719
页数:11
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