Enzymatic characteristics of cellobiose phosphorylase from Ruminococcus albus NE1 and kinetic mechanism of unusual substrate inhibition in reverse phosphorolysis

Biosci Biotechnol Biochem. 2012;76(4):812-8. doi: 10.1271/bbb.110954. Epub 2012 Apr 7.

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).

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Biocatalysis
  • Catalytic Domain
  • Cellobiose / metabolism*
  • Cloning, Molecular
  • Escherichia coli
  • Glucose / metabolism*
  • Glucosyltransferases / genetics
  • Glucosyltransferases / metabolism*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Monosaccharides / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Ruminococcus / chemistry
  • Ruminococcus / enzymology*
  • Substrate Specificity
  • Sugar Phosphates / metabolism*
  • Temperature

Substances

  • Bacterial Proteins
  • Monosaccharides
  • Recombinant Proteins
  • Sugar Phosphates
  • Cellobiose
  • Glucosyltransferases
  • cellobiose phosphorylase
  • Glucose