Modification of the ATP inhibitory site of the Ascaris suum phosphofructokinase results in the stabilization of an inactive T state.
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 microM 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., & Harris, B.G. (1987) J. Biol. Chem. 262, 14068-14073].(ABSTRACT TRUNCATED AT 250 WORDS)
Rao
G S
GS
Department of Biochemistry and Molecular Biology, Texas College of Osteopathic Medicine/University of North Texas, Fort Worth 76107.
Cook
P F
PF
Harris
B G
BG
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D001665:Binding Sites; D004220:Disulfides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D005635:Fructosediphosphates / Q000737:chemistry; D005636:Fructosephosphates / Q000737:chemistry; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology
D004220:Disulfides; D005635:Fructosediphosphates; D005636:Fructosephosphates; D011725:Pyridines; C007542:4,4'-dipyridyl disulfide; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1