Effector-induced conformational transitions in Ascaris suum phosphofructokinase. A fluorescence and circular dichroism study.
Results of activity and spectral studies using fluorescence and circular dichroism show that AMP and fructose 2,6-bisphosphate (F-2,6-P2) activate Ascaris suum phosphofructokinase through specific and similar conformational changes. Inorganic compounds like (NH4)2SO4 and KH2PO4 also induce structural alterations in the enzyme in a manner different from those caused by AMP and F-2,6-P2. The enzyme is activated by both AMP and F-2,6-P2, in 20 mM phosphate buffer, pH 6.6, with 0.2 mM ATP and 1 mM F-6-P. The Kact values for AMP and F-2,6-P2 are 25 +/- 3 microM and 1.5 +/- 0.2 microM, respectively. Both effectors quench enzyme tryptophan fluorescence in phosphate, pH 6.6, in a concentration-dependent manner. The Kd values determined from the decrease in emission intensity at 342 nm as a function of effector concentration are 24 +/- 3 microM for AMP and 1.00 +/- 0.15 microM for F-2,6-P2, in excellent agreement with the values of Kact. Both effectors also produce dramatic changes in the CD spectrum of the enzyme, in the region from 240 to 190 nm representing the peptide backbone. Secondary structure calculations suggest an increase in the alpha-helical content of the enzyme in the presence of either effector. The Kd values obtained from the concentration dependence of the decrease in ellipticity at 210 nm are 22.8 +/- 5.3 microM and 1.3 +/- 0.2 microM, respectively, for AMP and F-2,6-P2, once again in close agreement with the Kact values for these effectors. The data imply that activation of phosphofructokinase by these effectors is concomitant with structural changes in the enzyme. Further, comparison of the difference CD spectra for the effects of AMP and F-2,6-P2 show that both of them produce similar conformational changes and probably stabilize a similar final activated state of the enzyme. Other hexose phosphate analogues such as fructose 6-phosphate, glucose 1,6-bisphosphate, and fructose 1,6-bisphosphate do not affect the CD spectrum of the enzyme. Ammonium sulfate has no effect on the CD spectrum of the enzyme in phosphate buffer but does cause a significant alteration in the spectrum obtained in Mes. Gel filtration high performance liquid chromatography using a Borosil TSK 400 column shows that the tetrameric state of the native enzyme is not affected by the presence of the effectors.
Rao
G S
GS
Department of Biochemistry & Molecular Biology, Texas College of Osteopathic Medicine, Fort Worth 76107.
Cook
P F
PF
Harris
B G
BG
D000249:Adenosine Monophosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D002021:Buffers; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D005636:Fructosephosphates / Q000494:pharmacology; D007700:Kinetics; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
D002021:Buffers; D005636:Fructosephosphates; D000249:Adenosine Monophosphate; D014364:Tryptophan; D010732:Phosphofructokinase-1