Magnetic resonance studies of the interactions of Mn2+, substrates, and effectors with an allosteric pyruvate kinase from yeast (YPK)

Their early studies of the binding of Mn2+ and PEP to YPK used an enzyme that was partially proteolyzed. These studies were repeated with intact enzyme as judged by the subunit molecular weight of 57,000 and a specific activity of 340 units/mg. In the absence of substrates or effectors, only the weak binding of Mn2+ to YPK at multiple sites was detected. The presence of a saturating level of FDP (1.0 mM) or P-enolpyruvate (1.1 mM) or both, resulted in the substoichiometric binding of Mn2+ to 0.5 +/- 0.1 sites per subunit with a dissociation constant of 20 +/- 10 μM. The binding of Mn2+ at this site resulted in enhancement of its paramagnetic effect on 1/T1 of water protons by factors of 5.2 and 2.5 in the ternary FDP and P-enolpyruvate complexes respectively, and 1.9 in the quaternary complex. Titrations of mixtures of YPK (86 - 100 μM), and Mn2+ (60 μM) with P-enolpyruvate detected weak substrate binding (K/sub 0.5/ ∼ 1 mM) which became tighter by an order of magnitude (K/sub 0.5 ∼ 0.1 mM) in the presence of FDP. The binding of FDP to YPK and Mn2+ showed positive cooperativity with K/sub 0.5 ∼ 0.33 mM, which tightened to < 20 μM in the presence of P-enolpyruvate. The present data confirm and extend their early studies, and indicate half-site stoichiometry in the binding of the divalent cation activator