Kinetic studies of a recombinant cellobiose phosphorylase (CBP) of the Clostridium thermocellum YM4 strain expressed in Escherichia coli.
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 P(i) 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(i). In the synthetic reaction, the enzyme showed three times higher activity with beta-D-glucose than with alpha-D-glucose, and also showed weak activity with 1,5-anhydro-D-glucitol, indicating that the beta-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.
Kim
Yeon-Kye
YK
Enzyme Laboratory, National Food Research Institute, Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Kitaoka
Motomitsu
M
Krishnareddy
Manem
M
Mori
Yutaka
Y
Hayashi
Kiyoshi
K
D001665:Binding Sites; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D005964:Glucosyltransferases; C034714:cellobiose phosphorylase; D005947:Glucose