Influence of substrates and MgADP on the time-resolved intrinsic fluorescence of phosphofructokinase from Escherichia coli. Correlation of tryptophan dynamics to coupling entropy.
The influence of that MgADP and the substrate ligands MgATP and fructose 6-phosphate (Fru-6-P) have on the structure of E. coli phosphofructokinase (PFK) in the vicinity of the single tryptophan that exists in each subunit has been examined by employing both steady-state and time-resolved measurements of the tryptophan fluorescence. The accessibility of the tryptophan to iodide quenching is over 1 order of magnitude less than experienced by N-acetyltryptophanamide in solution but varies nonetheless with the state of ligation. Most, but not all, of these changes correlate with changes in the degree of local motion available to the tryptophan side chain as determined by steady-state and time-resolved polarization measurements. When the data obtained from differential polarization experiments are fit to a model in which the motion of the tryptophan side chain is able to move with high frequency within a cone of limited amplitude as part of an otherwise slowly tumbling spherical protein, it was found that ligands primarily affect the amplitude of the available local motion. By interpreting these effects with reference to the disproportionation equilibria which define the negative coupling free energy between MgADP and Fru-6-P and the positive coupling free energy between MgADP and MgATP, it is apparent that changes in the local motion amplitudes correlate with the sign of the component coupling entropy previously determined from van't Hoff analyses (Johnson & Reinhart, 1994).(ABSTRACT TRUNCATED AT 250 WORDS)
Johnson
J L
JL
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019.
Reinhart
G D
GD
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005454:Fluorescence Polarization; D007454:Iodides / Q000737:chemistry; D007700:Kinetics; D010732:Phosphofructokinase-1 / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D007454:Iodides; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1