Modification of the ATP inhibitory site of the Ascaris suum phosphofructokinase results in the stabilization of an inactive T state

• Published in: Biochemistry (Easton) 1991-10, Vol.30 (41), p.9998-10004
• Main Authors: Rao, G. S. Jagannatha, Cook, Paul F, Harris, Ben G
• Format: Article
• Language: English
• Subjects: 2',3'-dialdehyde ATP; 550200 - Biochemistry; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ADENOSINE TRIPHOSPHATE; Adenosine Triphosphate - pharmacology; ADENOSINTRIFOSFATO; Analytical, structural and metabolic biochemistry; Animals; ASCARIDAE; ASCARIS; Ascaris - enzymology; ASCARIS SUUM; ASCHELMINTHES; ATP; BASIC BIOLOGICAL SCIENCES; Binding Sites; BIOCHEMICAL REACTION KINETICS; Biological and medical sciences; CHROMATOGRAPHY; CONFORMATIONAL CHANGES; Disulfides - pharmacology; Enzyme Activation - drug effects; Enzyme Stability - drug effects; ENZYMES; Enzymes and enzyme inhibitors; Fructosediphosphates - chemistry; Fructosephosphates - chemistry; Fundamental and applied biological sciences. Psychology; HELMINTHS; HYDROGEN COMPOUNDS; INACTIVATION; INHIBICION; INHIBITION; KINETICS; LIQUID COLUMN CHROMATOGRAPHY; NEMATODES; NUCLEOTIDES; ORGANIC COMPOUNDS; PARASITES; Phosphofructokinase-1 - antagonists & inhibitors; Phosphofructokinase-1 - chemistry; Phosphofructokinase-1 - drug effects; PHOSPHORUS-GROUP TRANSFERASES; PHOSPHOTRANSFERASES; Protein Conformation - drug effects; PROTEINS; Pyridines - pharmacology; REACTION KINETICS; SEPARATION PROCESSES; TRANSFERASAS; TRANSFERASE; TRANSFERASES; TRITIUM COMPOUNDS; UPTAKE
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00105a026

Treatment of the Ascaris suum phosphofructokinase (PFK) with 2',3'-dialdehyde ATP (oATP) results in an enzyme form that is inactive. The conformational integrity of the active site, however, is preserved, suggesting that oATP modification locks the PFK into an inactive T state that cannot be activated. A rapid, irreversible first-order inactivation of the PFK is observed in the presence of oATP. The rate of inactivation is saturable and gives a KoATP of 1.07 +/- 0.27 mM. Complete protection against inactivation is afforded by high concentrations of ATP, and the dependence of the inactivation rate on the concentration of ATP gives a Ki of 326 +/- 26 micromole for ATP which is 22-fold higher than the Km for ATP at the catalytic site but close to the binding constant for ATP to the inhibitory site. Fructose 6-phosphate, fructose 2,6-bisphosphate, and AMP provide only partial protection against modification. The pH dependence of the inactivation rate gives a pKa of 8.4 +/- 0.1. Approximately 2 mol of [3H]oATP is incorporated into a subunit of PFK concomitant with 90% loss of activity, and ATP prevents the derivatization of 1 mol/subunit. The oATP-modified enzyme is not activated by AMP or fructose 2,6-bisphosphate. oATP has no effect on the activity of a desensitized form of PFK in which the ATP inhibitory site is modified with diethyl pyrocarbonate but with the active site intact [Rao, G. S. J., Wariso, B. A., Cook, P. F., Hofer, H. W., and Harris, B. G. (1987) J. Biol. Chem. 262, 14068-14073]. This desensitized enzyme incorporates only 0.2-0.3 mol of [3H]oATP/subunit, suggesting that in the native enzyme inactivation perhaps results from the modification of the ATP inhibitory site rather than the catalytic site. Modification of an active-site thiol by 4,4'-dithiodipyridine is prevented by ATP before and after oATP treatment. Finally, get filtration HPLC studies show that the oATP-modified enzyme retains its tetrameric state and neither the tryptophan fluorescense the circular dichroic spectra of the modified enzyme are affected by fructose 2,6-bisphosphate, suggesting that the enzyme is locked into a tetrameric inactive T state