Proton NMR Studies of the Paramagnetic Effects of Ni²⁺ and of CrATP on the Active Site Histidine Residues of Rabbit Pyruvate Kinase Type M₁

Shunsuke Meshitsuka

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

Six of the fourteen histidine residue of pyruvate kinase from rabbit muscle (molecular weight 237,000) yield resolved imidazole C-2 proton resonances. The divalent cation Ni²⁺ was found to bind specifically to the active site of pyruvate kinase in competition with Mg²⁺. Moreover, the presence of the substrate P-enolpyruvate greatly enhanced the effect of Ni²⁺ on the C-2 proton of his [3]. The paramagnetic substrate β,γ bidentate CrATP broadened all of the resolved histidine resonances. Specific displacement of CrATP from the active site by P-enolpyruvate resulted in the selective narrowing of the his [2] and his [3] resonances. This finding confirms the presence of his [3] at the active site and, further indicates the presence of his [2] at or near the metal-nucleotide binding region of the active site.

Key words: CrATP; histidine; NMR; pyruvate kinase

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