Expression of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli.
Chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was expressed in E. coli by using a pET3a T7 RNA polymerase-based expression system and was purified to homogeneity. The kinase and bisphosphatase of the expressed bifunctional enzyme had kinetic properties identical to those of the native chicken liver enzyme. However, the kinase activity of the chicken liver enzyme was 7-fold higher, while the bisphosphatase activity was 50 percent lower than those of the rat liver enzyme. Cys-256 of the rat liver bisphosphatase domain is not conserved in the chicken liver enzyme. A site-directed mutation was engineered at Cys-256 of the rat liver enzyme and the results indicate that the variation of this residue is not responsible for the difference in fructose-2,6-bisphosphatase activity between the rat and chicken liver enzymes. It is postulated that the difference in the kinase/bisphosphatase activity ratios of these two enzymes results from differences in their NH2-terminal regions.
Li
L
L
Shanghai Institute of Biochemistry, Academia Sinica, China.
Yao
W Z
WZ
Lange
A J
AJ
Pilkis
S J
SJ
Dong
M Q
MQ
Yin
Y
Y
Xu
G J
GJ
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003062:Codon; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D010770:Phosphotransferases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D003062:Codon; D017931:DNA Primers; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium