title: Glycine 122 is essential for cooperativity and binding of Mg2+ to porcine fructose-1,6-bisphosphatase.
abstract: Site-directed mutagenesis of an amino acid residue in the substrate binding site of porcine fructose-1,6-bisphosphatase was carried out based on the crystal structure of the enzyme (Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., and Lipscomb, W. N. (1993) Biochemistry 32, 1844-1857). A mutant enzyme form of fructose-1,6-bisphosphatase, G122A, was purified and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectrometry (CD), and initial rate kinetics. There were no discernible differences between the secondary structures of the wild-type and the mutant enzyme on the basis of CD data. Altering Gly-122 to alanine caused a significant decrease in the enzyme's activity and affinity for Mg2+. The kcat for this mutant enzyme was only about 5% of that of wild-type fructose-1,6-bisphosphatase, and the Ka for Mg2+ was about 20-fold higher than that of the wild-type enzyme. The Ki for AMP was increased 77-fold in the case of the mutant enzyme; however, the Hill coefficient was unaltered. Most importantly, it was observed that replacement of Gly-122 with alanine caused the total loss of cooperativity for Mg2+. It is concluded that Gly-122 is essential for Mg2+ cooperativity and important for binding of Mg2+ and AMP as well as for enzyme activity.
authors: Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
authors: R
authors: R
authors: Zhang
authors: L
authors: L
authors: Chen
authors: V
authors: V
authors: Villeret
authors: H J
authors: HJ
authors: Fromm
mesh_terms: D000818:Animals; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013552:Swine
chemical_list: D006597:Fructose-Bisphosphatase; D008274:Magnesium; D005998:Glycine