Kinetic studies on endo-beta-galactosidase by a novel colorimetric assay and synthesis of N-acetyllactosamine-repeating oligosaccharide beta-glycosides using its transglycosylation activity.
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.
Murata
Takeomi
T
Department of Applied Biological Chemistry, Shizuoka University, Japan. actmura@agr.shizuoka.ac.jp
Hattori
Takeshi
T
Amarume
Satoshi
S
Koichi
Akiko
A
Usui
Taichi
T
D000606:Amino Sugars / Q000737:chemistry*; D001441:Bacteroides fragilis / Q000201:enzymology; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D002240:Carbohydrate Sequence; D016971:Citrobacter freundii / Q000201:enzymology; D006026:Glycoside Hydrolases; D006027:Glycosides / Q000096:biosynthesis* / Q000737:chemistry*; D006031:Glycosylation; D006868:Hydrolysis; D007536:Isomerism; D007632:Keratan Sulfate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry*; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry* / Q000378:metabolism*
D000606:Amino Sugars; D006027:Glycosides; D009596:Nitrophenols; D009844:Oligosaccharides; C000458:N-acetyllactosamine; D007632:Keratan Sulfate; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase