TY - JOUR
T1 - Site-directed mutants of rat testis fructose 6-phosphate, 2- kinase/fructose 2,6-bisphosphatase
T2 - Localization of conformational alterations induced by ligand binding
AU - Helms, Michael K.
AU - Hazlett, Theodore L.
AU - Mizuguchi, Hiroyuki
AU - Hasemann, Charles A.
AU - Uyeda, Kosaku
AU - Jameson, David M.
PY - 1998/10/6
Y1 - 1998/10/6
N2 - 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 μM, respectively. These observations are considered in light of the recently published crystal structure for this bifunctional enzyme.
AB - 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 μM, respectively. These observations are considered in light of the recently published crystal structure for this bifunctional enzyme.
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U2 - 10.1021/bi980202w
DO - 10.1021/bi980202w
M3 - Article
C2 - 9760241
AN - SCOPUS:0032491173
SN - 0006-2960
VL - 37
SP - 14057
EP - 14064
JO - Biochemistry
JF - Biochemistry
IS - 40
ER -