ACID-BASE CATALYTIC MECHANISM AND PH-DEPENDENCE OF FRUCTOSE 2,6-BISPHOSPHATE ACTIVATION OF THE ASCARIS-SUUM PHOSPHOFRUCTOKINASE

A form of phosphofructokinase (PFK) from Ascaris suum desensitized to hysteresis in the reaction time course and ATP allosteric inhibition h as been used to study the activation by fructose 2,6-bisphosphate (F26 P(2)) at varied pH in both reaction directions. In the direction of ph osphorylation of F6P, V and V/K-MgATP are constant over the pH range 6 -9, while V/K-F6P decreases at low pH, giving a pK value of 7.0, and a t high pH, giving a pK of 8.9. V and V/K-MgATP are insensitive to the presence of F26P(2), but V/K-F6P is increased by a constant amount in the presence of saturating F26P(2) over the entire pH range studied. T he concentration of F26P(2) that gives half the change in V/K-F6P, K-a ct, increases as the pH decreases, giving a pK of 7.4, reflecting an e nzyme group that must be unprotonated for optimum binding of F26P(2). In the direction of phosphorylation of MgADP, V and V/K-MgADP are pH-i ndependent, and both are insensitive to the presence of F26P(2). V/K-F BP decreases at high pH, giving a pK of about 7.3, and is increased by a constant amount in the presence of F26P(2) over the entire pH range studied. A mechanism consistent with the data requires an enzymic gen eral base with a pK of 7.0 to accept a proton from the 1-hydroxyl of F 6P concomitant with nucleophilic attack of the hydroxyl on the gamma-p hosphate of MgATP, while a second enzyme group with a pK of 8.9 must b e protonated and is postulated either to neutralize the negative charg e on the gamma-phosphate of MgATP to facilitate the nucleophilic attac k or to bind the 6-phosphate of F6P. A group with a pK of 7.4 in the F 26P(2) binding site must be unprotonated for optimum binding of the ef fector and likely hydrogen-bonds to the hydroxyl group at C1, C2, or C 3 of F26P(2). The effect of F26P(2) is the pH-independent decrease of the off rate for F6P and FBP from binary and ternary enzyme-reactant c omplexes.