Site-directed mutants of rat testis fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase: localization of conformational alterations induced by ligand binding.
Site-directed mutagenesis was utilized to construct mutants, containing one or two tryptophan residues, of the bifunctional enzyme fructose 6-phosphate,2-kinase-fructose 2,6-bisphosphatase. Two of the single-tryptophan mutants (W15 and W64) had the tryptophan residue located in the kinase domain, which is in the N-terminal half, and two (W299 and W320) had the tryptophan residue located in the phosphatase domain, which is in the C-terminal half. The double-tryptophan mutants were W15/W64, W15/W299, W64/W299, and W299/W320. Dynamic polarization data indicated that these tryptophan residues had varying degrees of local mobility. Steady-state polarization data revealed energy transfer between the tryptophan residues in the double mutant W299/W320 but not in the W15/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 did not affect the anisotropies of any of the other single-tryptophan mutants. Binding of fructose-2,6-bisphosphate also significantly increased 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, which suggests that the N-terminus is located near the kinase active site. These increases in anisotropies were used to estimate the dissociation constants of fructose-6-phosphate and fructose-2,6-bisphosphate, which were 29 +/- 3 and 2.1 +/- 0.3 microM, respectively. These observations are considered in light of the recently published crystal structure for this bifunctional enzyme.
Department of Genetics and Molecular Biology, University of Hawaii at Manoa, Honolulu 96822, USA.
M K
MK
Helms
T L
TL
Hazlett
H
H
Mizuguchi
C A
CA
Hasemann
K
K
Uyeda
D M
DM
Jameson
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D005454:Fluorescence Polarization; D005635:Fructosediphosphates / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D008024:Ligands; D008297:Male; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics*; D010770:Phosphotransferases / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D013737:Testis / Q000201:enzymology*; D014364:Tryptophan / Q000235:genetics
D005635:Fructosediphosphates; D005636:Fructosephosphates; D008024:Ligands; D009097:Multienzyme Complexes; D010649:Phenylalanine; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D014364:Tryptophan; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases