STRUCTURAL ASPECTS OF THE ALLOSTERIC INHIBITION OF FRUCTOSE-1,6-BISPHOSPHATASE BY AMP - THE BINDING OF BOTH THE SUBSTRATE-ANALOG 2,5-ANHYDRO-D-GLUCITOL 1,6-BISPHOSPHATE AND CATALYTIC METAL-IONS MONITORED BY X-RAY CRYSTALLOGRAPHY

The crystal structures of the T form pig kidney fructose-l,6-bisphosphatase (EC 3.1.3.11) complexed with AMP, the substrate analogue 2,5-anhydro-D-glucitol 1,6-bisphosphate (AhG-1,6-P-2), and Mn2+ at concentrations of 5, 15, 100, and 300 mu M have been determined and refined at resolutions of 2.1-2.3 Angstrom to R factors which range from 0.180 to 0.195, respectively. Two metal ions per active site have been identified, one at a binding site of high affinity (metal site 1'), the second in a low affinity site (metal site 2'). The l-phosphate group of the substrate analogue coordinates to the metal ion at site 1', but not at site 2'. In these four complexes, the distances between the two metal ions are all within 0.2 Angstrom of 4.3 Angstrom. In the previously determined R form structure of Fru-1,6-Pase complexed with AhG-1,6-P-2 and Mn2+, there are also two metal ions in the active site at metal sites 1 and 2. The metal ion at site 1 is only 0.6 Angstrom displaced from the metal ion at site 1' in the T form and is also coordinated to the l-phosphate group of AhG-1,6-P-2. However, the second metal ion is located in two distinct sites which are 1.4 Angstrom apart in the T and R form structures. In the R form the Mn2+ at site 2 is coordinated to the 1-phosphate group of the substrate analogue. This metal ion is apparently required to orient the phosphate group for nucleophilic attack at the phosphorus center. We demonstrate here that differences between metal binding sites in the T and R forms are central to a structural explanation for the allosteric inhibition of Fru-1,6-Pase by AMP when two Mn2+ or two Zn2+ are required for catalytic activity. Upon binding of AMP, the AMP domain (residues 1-200) is rotated 1.9 degrees relative to the FBP domain (residues 200-335). This movement affects the positions of most residues involved in metal binding. Thus in the T form these conformational changes observed for these residues prevent the binding of Mn2+ ion at site 2. The metal ion found at site 2' (T form) is not coordinated to the l-phosphate group of the substrate, which is no longer well oriented for the nucleophilic attack at the phosphorus center.