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]. © 1993 Academic Press, Inc.