Proton NMR and Relaxation Studies of Pyruvate Kinase M₁ from Rabbit Muscle

Shunsuke Meshitsuka

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

The rotational correlation times of pyruvate kinase M₁ (3.5-5.7 ns) are an order of magnitude smaller than the tumbling time of the entire protein (56 ns). The divalent cation Ni²⁺ was found to bind specifically to the active site of pyruvate kinase in competition with Mn²⁺ with a 1.7-fold greater affinity but with a 4-fold lower V_max than with Mg²⁺ and with a 7.7-fold lower V_max than with Mg²⁺. The binding of the paramagnetic Ni²⁺ ion to the active site of pyruvate kinase selectively increased the longitudinal (1/T₁) and transverse (1/T₂) relaxation rates of one of the resolved histidine C-2 resonances (his[3]) with much smaller effects on the others. The distance from the bound Ni²⁺ to the imidazole C-2 proton of his [3] calculated from the 1/T₁ value of this proton (6 Å) is greater than expected for direct coordination suggesting a second sphere Ni-imidazole complex. The binding of the substrate P-enolpyruvate greatly enhanced the effect of Ni²⁺ on 1/T₂ of the his [3] resonance suggesting a stronger interaction of Ni²⁺ with his [3] in the P-enolpyruvate complex.

Key words: nuclear magnetic resonance; spin lattice relaxation time; spin spin relaxation time; pyruvate kinase

RETURN