Expression of bovine heart fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase and determination of the role of the carboxyl terminus by mutagenesis.
Bovine heart fructose 6-P,2-kinase:fructose 2,6-bisphosphatase was expressed in Escherichia coli. In order to determine the role of the carboxyl-terminal peptide, 49 and 78 amino acids from the C-terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type and mutant enzymes were purified to homogeneity, and the steady-state kinetics of the mutant enzymes were compared to those of the wild-type enzyme. Deletion of 49 residues (Del 49) resulted in a 35% decrease in KmFru6P, a 36% increase in Vmax, and a 2-fold increase in Kcat/Km of the kinase. There was no change in the kinetic properties of the phosphatase activity. Deletion of 78 residues (Del 78) resulted in a 4.5-fold decrease in KmFru6P, a 2.5-fold increase in Vmax, a 12-fold increase in kcat/Km of the kinase, and a 3-fold increase in kcat/Km of the phosphatase. Phosphorylation of the wild-type and Del 49 enzymes resulted in decreased KmFru6P and activation of the kinase without affecting the phosphatase activity. Thermal inactivation rates of the wild-type and Del 49 enzymes were similar, but the rate of Del 78 was more rapid. The phosphorylated wild-type and Del 49 enzymes were more sensitive to thermal inactivation than the dephospho forms. Urea inactivation of the kinase and phosphatase of wild-type and Del 49 were similar, but Del 78 was more sensitive to urea. All phosphorylated enzymes were more susceptible to urea inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)
Abe
Y
Y
Department of Veterans Affairs Medical Center, Dallas, TX 75216.
Minami
Y
Y
Li
Y
Y
Nguyen
C
C
Uyeda
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D014508:Urea / Q000494:pharmacology
D017931:DNA Primers; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D014508:Urea; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases