Kinetic Studies of a Recombinant Cellobiose Phosphorylase (CBP) of the Clostridium thermocellum YM4 Strain Expressed in Escherichia coli

• Published in: Journal of biochemistry (Tokyo) 2002-08, Vol.132 (2), p.197-203
• Main Authors: Kim, Yeon-Kye, Kitaoka, Motomitsu, Krishnareddy, Manem, Mori, Yutaka, Hayashi, Kiyoshi
• Format: Article
• Language: English
• Subjects: anomeric hydroxyl group; Binding Sites; cellobiose phosphorylase; Cloning, Molecular; Clostridium - enzymology; Clostridium - genetics; Clostridium thermocellum YM4; competitive substrate inhibition; Escherichia coli - genetics; Escherichia coli - metabolism; Glucose - metabolism; Glucosyltransferases - antagonists & inhibitors; Glucosyltransferases - genetics; Glucosyltransferases - isolation & purification; Glucosyltransferases - metabolism; Kinetics; ordered bi bi mechanism; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Substrate Specificity
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a003210

A cellobiose phosphorylase (CBP) cloned from the Clostridium thermocellum YM4 strain was purified to homogeneity, and the reaction mechanisms of both the phosphorolytic and synthetic reactions were studied in detail. The enzyme reaction proceeded via an ordered bi bi mechanism, in which P1 bound to the enzyme prior to D-cellobiose and then G 1-P was released after D-glucose. The order of substrate binding was different from that of CBP from Cellvibrio gilvus, which bound to cellobiose prior to P,. In the synthetic reaction, the enzyme showed three times higher activity with p-D-glucose than with a-D-glucose, and also showed weak activity with 1, 5-anhydro-D-glucitol, indicating that the p-anomeric hydroxyl group of D-glucose is highly required. However, even when it is removed enzyme activity remains. The substrate specificity and kinetic studies revealed that the configurations of the C3 and C4 hydroxyl groups were strictly required for the enzyme activity, whereas those of C2 and C6 could be substituted or deleted. The mechanism of substrate inhibition by D-glucose was studied in detail and it was concluded that D-glucose competed with G 1-P for its binding site in the synthetic reaction.