Kinetic characterization of a T-state of Ascaris suum phosphofructokinase with heterotropic negative cooperativity by ATP eliminated

The affinity analogue, 2',3'-dialdehyde ATP has been used to chemically mod ify the ATP-inhibitory site of Ascaris suum phosphofructokinase, thereby lo cking the enzyme into a less active T-state. This enzyme form has a maximum velocity that is 10% that of the native enzyme in the direction of fructos e g-phosphate (F6P) phosphorylation. The enzyme displays sigmoid saturation for the substrate fructose 6-phosphate (S-0.5 (F6P) = 19 mM and n(H) = 2.2 ) at pH 6.8 and a hyperbolic saturation curve for MgATP with a K-m identica l to that for the native enzyme. The allosteric effecters, fructose 2,6-bis phosphate and AMP, do not affect the S-0.5 for F6P but produce a slight (1. 5- and 2-fold, respectively) V-type activation with K-a values (effector co ncentration required for half-maximal activation) of 0.40 and 0.24 mM, resp ectively. Their activating effects are additive and not synergistic. The ki netic mechanism for the modified enzyme is steady-state-ordered with MgATP as the first substrate and MgADP as the last product to be released from th e enzyme surface. The decrease in V and V/K values for the reactants likely results from a decrease in the equilibrium constant for the isomerization of the E:MgATP binary complex, thus favoring an unisomerized form. The V an d V/K-F6P are pH dependent with similar pK values of about 7 on the acid si de and 9.8 on the basic side. The microenvironment of the active site appea rs to be affected minimally as evidenced by the similarity of the pK values for the groups involved in the binding site for F6P in the modified and na tive enzymes. (C) 1999 Academic Press.