Kinetic Characterization of a T-State ofAscaris suumPhosphofructokinase with Heterotropic Negative Cooperativity by ATP Eliminated

The affinity analogue, 2′,3′-dialdehyde ATP has been used to chemically modify the ATP-inhibitory site ofAscaris suumphosphofructokinase, thereby locking 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 fructose 6-phosphate (F6P) phosphorylation. The enzyme displays sigmoid saturation for the substrate fructose 6-phosphate (S0.5(F6P) = 19 mM andnH= 2.2) at pH 6.8 and a hyperbolic saturation curve for MgATP with aKmidentical to that for the native enzyme. The allosteric effectors, fructose 2,6-bisphosphate and AMP, do not affect theS0.5for F6P but produce a slight (1.5- and 2-fold, respectively)V-type activation withKavalues (effector concentration required for half-maximal activation) of 0.40 and 0.24 mM, respectively. Their activating effects are additive and not synergistic. The kinetic 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 the enzyme surface. The decrease inVandV/Kvalues 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. TheVandV/KF6Pare pH dependent with similar pKvalues of about 7 on the acid side and 9.8 on the basic side. The microenvironment of the active site appears to be affected minimally as evidenced by the similarity of the pKvalues for the groups involved in the binding site for F6P in the modified and native enzymes.