Site-specific mutagenesis of the metal binding sites of porcine fructose-1,6-bisphosphatase.
Mutations in the metal binding sites of porcine fructose-1,6-bisphosphatase were carried out by site-specific mutagenesis based on the crystal structure of the enzyme. The mutant and wild-type enzymes have been characterized by circular dichroism spectrometry and initial-rate kinetics. One of the mutant forms of fructose-1,6-bisphosphatase (Glu280Gln) is associated with a single metal site, whereas two other mutants (Glu97Gln and Asp118Asn) ligate two metal ions. The mutant enzymes exhibit very large decreases in kcat relative to the wild-type enzyme; however, other kinetic parameters, such as Km values, are not greatly altered. Metal binding cooperativity and binding affinity is decreased in the mutants compared to wild-type fructose-1,6-bisphosphatase. Mutations in the metal binding sites greatly enhance the enzyme's affinity for AMP, a potent regulator of fructose-1,6-bisphosphatase activity. The results of these investigations are fully consistent with predictions made on the role of specific amino acid residues at the metal binding sites in porcine fructose-1,6-bisphosphatase from X-ray diffraction studies [Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., and Lipscomb, W.N. (1993) Biochemistry, 32, 1844-1857].
Chen
L
L
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Hegde
R
R
Chen
M
M
Fromm
H J
HJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006597:Fructose-Bisphosphatase / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013552:Swine; D015032:Zinc / Q000378:metabolism
D017931:DNA Primers; D008670:Metals; D011994:Recombinant Proteins; D008345:Manganese; D006597:Fructose-Bisphosphatase; D008274:Magnesium; D015032:Zinc