title: Enzymatic characteristics of cellobiose phosphorylase from Ruminococcus albus NE1 and kinetic mechanism of unusual substrate inhibition in reverse phosphorolysis.
abstract: Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose to produce α-D-glucopyranosyl phosphate (Glc1P) and D-glucose. It is an essential enzyme for the metabolism of cello-oligosaccharides in a ruminal bacterium, Ruminococcus albus. In this study, recombinant R. albus CBP (RaCBP) produced in Escherichia coli was characterized. It showed highest activity at pH 6.2 at 50 °C, and was stable in a pH range of 5.5-8.8 and at below 40 °C. It phosphorolyzed only cellobiose efficiently, and the reaction proceeded through a random-ordered bi bi mechanism, by which inorganic phosphate and cellobiose bind in random order and D-glucose is released before Glc1P. In the synthetic reaction, RaCBP showed highest activity to D-glucose, followed by 6-deoxy-D-glucose. D-Mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, 1,5-anhydro-D-glucitol, and gentiobiose also served as acceptors, although the activities for them were much lower than for D-glucose. D-Glucose acted as a competitive-uncompetitive inhibitor of the reverse synthetic reaction, which bound not only the Glc1P site (competitive) but also the ternary enzyme-Glc1P-D-glucose complex (uncompetitive).
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Hamura
authors.forename: Wataru
authors.initials: W
authors.lastname: Saburi
authors.forename: Shotaro
authors.initials: S
authors.lastname: Abe
authors.forename: Naoki
authors.initials: N
authors.lastname: Morimoto
authors.forename: Hidenori
authors.initials: H
authors.lastname: Taguchi
authors.forename: Haruhide
authors.initials: H
authors.lastname: Mori
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Matsui
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D002475:Cellobiose / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D005947:Glucose / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009005:Monosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D045857:Ruminococcus / Q000737:chemistry / Q000201:enzymology*; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D009005:Monosaccharides; D011994:Recombinant Proteins; D013403:Sugar Phosphates; D002475:Cellobiose; D005964:Glucosyltransferases; C034714:cellobiose phosphorylase; D005947:Glucose