Yonago Acta medica 1996;37:205-212

Chemical Modification and Nuclear Magnetic Resonance Studies of the Adenosine 5'-Triphosphate Binding Site of Pyruvate Kinase M1

Shunsuke Meshitsuka and Takayuki Nose

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

The imidazole C-2 proton NMR signals of six of the fourteen histidine residues per subunit of rabbit muscle pyruvate kinase are resolved. Titration with two to three equivalents of diethylpyrocarbonate completely modifies his [2] and to a lesser extent his [1], as monitored by decreases in the intensity of their respective NMR signals and the parallel appearance of new peaks due to ethoxycarbonyl histidine, but it does not significantly modify other residues. Such chemical modification results in the complete loss of enzymatic activity in the overall phosphoryl transfer reaction. However the phosphate-stimulated enolization of pyruvate remains the activity, indicating that only the MgATP binding region of the active site has been affected by the modification of his [2]. Independent evidence for the presence of his [2] at the MgATP binding site of pyruvate kinase is provided by a specific intermolecular negative NOE on the adenine H-2 proton of MgATP observed upon selective pre-irradiation of the C-2 proton of his [2]. No effect is observed upon pre-irradiation of the C-4 proton of his [2] and no other specific inter-molecular NOEs are detected. These results establish the presence of his [2] at the active site of pyruvate kinase, in close proximity to the adenine ring of the bound MgATP substrate.

Key words: chemical modification; diethylpyrocarbonate; nuclear magnetic resonance; nuclear Overhauser effect; pyruvate kinase

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