Structure of the oxalate-ATP complex with pyruvate kinase: ATP as a bridging ligand for the two divalent cations

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Abstract
The 2 equiv of divalent cation that are required cofactors for pyruvate kinase reside in sites of different affinities for different species of cation [Baek, Y.H., and Nowak, T. (1982) Arch. Biochem. Biophys. 217, 491-497]. The intrinsic selectivity of the protein-based site for Mn(II) and of the nucleotide-based site for Mg(II) has been exploited in electron paramagnetic resonance (EPR) investigations of ligands for Mn(II) at the protein-based site. Oxalate, a structural analogue of the enolate of pyruvate, has been used as a surrogate for the reactive form of pyruvate in complexes with enzyme, Mn(II), Mg(II), and ATP. Addition of Mg(II) to solutions of enzyme, Mn(II), ATP, and oxalate sharpens the EPR signals for the enzyme-bound Mn(II). Superhyperfine coupling between the unpaired electron spin of Mn(II) and the nuclear spin of 17O, specifically incorporated into oxalate, shows that oxalate is bound at the active site as a bidentate chelate with Mn(II). Coordination of the .gamma.-phosphate of ATP to this same Mn(II) center is revealed by observation of superhyperfine coupling from 17O regiospecifically incorporated into the .gamma.-phosphate group of ATP. By contrast, 17O in the .alpha.-phosphate or in the .beta.-phosphate groups of ATP does not influence the spectrum. Experiments in 17O-enriched water show that there is also a single water ligand bound to the Mn(II). These data indicate that ATP bridges Mn(II) and Mg(II) at the active site. A close spacing of the two divalent cations is also evident from the occurrence of magnetic interactions for complexes in which 2 equiv of Mn(II) are present at the active site. The structure for the enzyme.sbd.Mn(II).sbd.oxalate.sbd.Mg(II).sbd.ATP complex suggests a scheme for the normal reverse action of pyruvate kinase in which the divalent cation at the protein-based site activates the keto acid substrate through chelation and promotes phospho transfer by simultaneous coordination to the enolate oxygen and to a pendant oxygen from the .gamma.-phosphate of ATP.

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