TAILIEUCHUNG - Báo cáo khoa học: Kinetic studies on endo-b-galactosidase by a novel colorimetric assay and synthesis of N -acetyllactosamine-repeating oligosaccharide b-glycosides using its transglycosylation activity

Novel chromogenic substrates for endo-b-galactosidase were designed on the basis of the structural features of keratan sulfate. Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-pNP (2),which consists of two repeating units ofN-acetyl-lactosamine,was synthesized enzymatically by consecutive additions of GlcNAc and Gal residues top-nitrophenyl b-N-acetyllactosaminide. In a similar manner,Glc-NAcb1-3Galb1-4GlcNAcb-pNP (1),GlcNAcb1-3Galb1-4Glcb-pNP (3),Galb1-4GlcNAcb1-3Galb1-4Glcb-pNP (4), Galb1-3GlcNAcb1-3Galb1-4Glcb-pNP (5),and Galb1-6GlcNAcb1-3Galb1-4Glcb-pNP (6) were synthesized as analogues of2. Endo-b-galactosidases released GlcNAcb-pNP or Glcb-pNP in an endo-manner from each substrate | Eur. J. Biochem. 270 _709 _7 9 2003 FEBS 2003 doi Kinetic studies on endo-p-galactosidase by a novel colorimetric assay and synthesis of V-acetyllactosamine-repeating oligosaccharide p-glycosides using its transglycosylation activity Takeomi Murata Takeshi Hattori Satoshi Amarume Akiko Koichi and Taichi Usui Department of Applied Biological Chemistry Shizuoka University Japan Novel chromogenic substrates for endo-b-galactosidase were designed on the basis of the structural features of keratan sulfate. Galp1-4GlcNAcf 1-3Galp1-4GlcNAcf -pNP 2 which consitss of two repeating uniss of N-acetyl-lactosamine was fynthesieed enyymaiícally by conrccuiire additions of GlcNAc and Gal residues to p-nitrophenyl b-N-acvtyllactosaminide. In a similar manner Glc-NAcp1-3Galp1-4GlcNAcp-pNP 1 GNNAcp1-3Gdp1-4Glcb-pNP 3 Gjalb1-4GlcNNcb1-3Galb1-4Gicb-pNP 4 Gv1P1-3G1cNNcP1-3gv1P1-4G1cP-pNP 5 and Gal 31-6GlcNNcb1-3Galb1-4Glcb-pNP 6 were synthesized as analogues of 2. Endo-b-galactosidases released GlcNNcb-pNP or Glcb-pNP in an endo-manner from each substrate. A colorimetric assay for endo-b-galactosidase was developed using the synthetic substrates on the basis of the determination of p-nitrophenol liberated from GlcNNcb-pNP or Glcb-pNP formed by the enzyme through a coupled reaction involving b-N-acvtylhexosaminidase b-NNHase or b-D-glucosidase. Kinetic analysis by this method showed that the value of Tmax Km of 2 for Escherichia freundii endo-b-galactosidase was higher than that for keratan sulfate indicating that 2 is very suitable as a sensitive substrate for analytical use in an endo-b-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-nitro-phenyl group. This was the same in the case of a .

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