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

概要

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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 Vmax than with Mg2+ and with a 7.7-fold lower Vmax 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.