SITE-DIRECTED MUTANTS OF RAT TESTIS FRUCTOSE 6-PHOSPHATE, 2-KINASE FRUCTOSE 2,6-BISPHOSPHATASE - LOCALIZATION OF CONFORMATIONAL ALTERATIONSINDUCED BY LIGAND-BINDING/

Site-directed mutagenesis was utilized to construct mutants, containin g one or two tryptophan residues, of the bifunctional enzyme fructose 6-phosphate,2-kinase-fructose 2,6-bisphosphatase. Two of the single-tr yptophan mutants (W15 and W64) had the tryptophan residue located in t he kinase domain, which is in the N-terminal half, and two (W299 and W 320) had the tryptophan residue located in the phosphatase domain, whi ch is in the C-terminal half. The double-tryptophan mutants were W15/W 64, W15/W299, W64/W299, and W299/W320. Dynamic polarization data indic ated that these tryptophan residues had varying degrees of local mobil ity. Steady-state polarization data revealed energy transfer between t he tryptophan residues in the double mutant W299/W320 but not in the W 15/W64, W15/W299, or W64/W299 mutants, indicating the proximity of the W299 and W320 residues. The binding of fructose-6-phosphate resulted in a significant increase in the anisotropy of the W15 mutants, but di d not affect the anisotropies of any of the other single-tryptophan mu tants. Binding of fructose-2,6-bisphosphate also significantly increas ed the anisotropy of W15. In the case of fructose-6-phosphate binding, the increased anisotropy was shown to be due to a restriction of the tryptophan residue's local mobility in the presence of bound ligand, w hich suggests that the N-terminus is located near the kinase active si te. These increases in anisotropies were used to estimate the dissocia tion constants of fructose-6-phosphate and fructose-2,6-bisphosphate, which were 29 +/- 3 and 2.1 +/-: 0.3 mu M, respectively. These observa tions are considered in light of the recently published crystal struct ure for this bifunctional enzyme.