Proton NMR Studies of Pyruvate Kinase Type M₂ from Rat Yoshida Ascites Hepatoma Cells and its Allosteric Effector Fructose-1,6-Biphosphate Complex

Shunsuke Meshitsuka

Department of Public Health, Faculty of Medicine, Tottori University, Yonago 683, Japan

Pyruvate kinase type M₂, in which activity was regulated allosterically, was purified from Yoshida ascites hepatoma cells of rats. Three of ten histidine residues were observed in the NMR spectra at 500 MHz. The pK' values of two histidine residues were obtained by pH titration in the NMR spectra both in the presence and absence of the allosteric effector Fru-1,6-P₂. The pK' were not changed by the binding of the effector. One of the histidine C-2 protons exhibited an upfield shift with the addition of the allosteric effector. The dissociation constants of Fru-1,6-P₂ were obtained by NMR titration at pH 7.4 and 5.9. The effector bound tightly at both pH values. An allosteric transition of sigmoidal to hyperbolic kinetics was observed at pH 7.5. However at pH 6.0, neither the interaction coefficient nor the K_m value was altered by the addition of the effector. The upfield shift of the histidine signal exhibited a conformational change induced by the allosteric effector.

Key words: allosteric effect; alternative splicing; isozyme; nuclear magnetic resonance; pyruvate kinase

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