Magnetic resonance studies on manganese-activated phosphofructokinase.

Abstract

Rabbit skeletal‐muscle phosphofructokinase exhibits similar kinetic behaviour and specific activity when manganese is used as the essential divalent cation in place of magnesium. At pH 7.5, the kinetics are Michaelis‐Menten with respect to fructose 6‐phosphate, ATP and manganous ion concentrations; the reaction, as a function of metal ion concentration, is inhibited competitively by excess free ATP and by calcium ions. Between pH 7.0 and 6.5, in the presence of high ATP concentrations, there is a sharp transition to sigmoid kinetics as indicated by an increase of the Hill interaction coefficient from 1 to 4.1. Preincubation of the enzyme with ligands has a marked effect on activity, particularly free divalent metals which cause dissociation and an almost total loss of activity.Studies on the rates of water‐proton longitudinal relaxation at pH 7.5 and 35 MHz indicate that, in the absence of nucleotides, the phosphofructokinase‐manganese system shows low relaxation enhancements (ɛb∼ 2). On titrating with ATP at fixed enzyme and manganese ion concentrations, the enhancement rises sharply, then falls again at very high ATP levels. An analysis of enhancement data at fixed ATP concentrations as a function of enzyme and metal ion concentrations, using a combination of graphical and computational methods, indicates that the protomer of 90000 molecular weight has 2 Mn‐ATP sites with dissociation constant 240 μM and an enhancement for the ternary complex ɛt, of 18. Free ATP binds competitively with a dissociation constant of 3 mM. Line‐broadening studies on the H‐2 and H‐8 resonances of the adenine ring in the high resolution spectrum of ATP reveal that the proton‐manganese distances in Mn‐ATP are almost unchanged in forming the ternary complex with enzyme. The kinetic and magnetic resonance data indicate that Mn‐ATP is the true substrate, which probably binds to the enzyme via a nucleotide bridge.