TOWARD THE ALLOSTERIC MECHANISM OF FRUCTOSE-1,6-BISPHOSPHATASE: X-RAY STRUCTURES AND KINETICS OF MUTANT ENZYMES. Boguslaw Stec, Guqiang Lu, Reimar Abraham, Eugene Giroux, Mark K. Williams, Evan R. Kantrowitz, Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, MA 02167.
Fructose-1,6-bisphosphatase (Fru-1,6-P2ase, E.C. 3.1.3.11) is a tetrameric enzyme playing an important role in a metabolic control of gluconeogenesis. Each identical subunit has 337 amino acid residues and a molecular weight of about 36,500. The physiological regulators AMP and Fru-1,6-P2 negatively modulate enzyme activity and, in reciprocal fashion, positively affect the activity of phosphofructokinase, a control point in glycolysis.
Fru-1,6-P2ase from pig kidney was cloned and expressed in E. coli. A series of mutant enzymes were created in order to study the allosteric and catalytic mechanism of its regulation. Mutant proteins were isolated, kinetically characterized, and crystallized in the T and R-allosteric states. Protein in the T state usually crystallized in P21212 space group with cell dimensions a= 61Å, b=166Å, c=79Å, and the R-state in P3221 space group with cell dimensions a=b=131Å, c=66Å in a hexagonal lattice.
The mutants: Arg243'Ala, Lys42'Ala and Arg22'Ala were prepared by site-specific mutagenesis and kinetically characterized. The mutant enzymes were isolated and crystallized. Data were collected on the Hamlin Are Detector to the resolution of 2Å, 2.2Å and 2.7Å, respectively. Structures were refined by X-plor and characterized.
Preliminary interpretation of the correlation between the kinetic results and the X-ray structures will be presented. These data suggest that the Arg243 is important for the proper inhibition by Fru-2,6-P2, Arg22 is important for the T-state stabilization and Lys42 for the transmission of the allosteric signal from the AMP binding site to the catalytic site which are more than 20Å apart.