Quantification of allosteric influence of Escherichia coli phosphofructokinase by frequency domain fluorescence.
The allosteric properties of the wild-type Escherichia coli phosphofructokinase were compared to the E187A mutant by using frequency-domain techniques. Tryptophan-shifted mutants comprising of double (W311Y/Y55W and W/311F/F188W) and triple (W311Y/Y55W/E187A and W311F/F188W/E187A) amino acid residue changes, which allowed for better fluorescence probing at targeted sites, were also compared to the wild-type and E187A. The additive nature of multiple mutations allowed one to partition the net effect of modifying residue 187. In general, the mutant enzymes displayed greater heterogeneity in sub-state population than did the wild-type enzyme. The semi-cone angle model was used to quantify the extent of depolarization of the fluorophore. Use of the model presupposes that the extent of depolarization directly correlates with the degree of flexibility of the fluorophore. A relationship has been established between the values determined from the semi-cone angle calculations and the thermodynamic components responsible for the allosteric linkage between the regulatory and substrate binding. Coupling interactions giving rise to positive entropy components are manifested by increasing flexibility of the ternary complexes rather than the binary complexes.
Pham
Audrey S
AS
Division of Pathology and Laboratory Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. aspham@mail.mdanderson.org
Reinhart
Gregory D
GD
D016880:Anisotropy; D003198:Computer Simulation; D004926:Escherichia coli / Q000201:enzymology*; D005583:Fourier Analysis; D006207:Half-Life; D007536:Isomerism; D008956:Models, Chemical; D008968:Molecular Conformation; D009154:Mutation; D025221:Phosphofructokinases / Q000032:analysis / Q000737:chemistry* / Q000145:classification*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013223:Statistics as Topic; D014364:Tryptophan / Q000032:analysis / Q000737:chemistry*
D014364:Tryptophan; D025221:Phosphofructokinases