Arrangement and conformations of substrates at the active site of pyruvate kinase from model building studies based on magnetic resonance data.

Seventeen distances from two paramagnetic reference points, as determined by nuclear relaxation studies of six active complexes of rabbit muscle pyruvate kinase, have been used to construct molecular models of two composite enzyme complexes. In the model of the hypothetical pyruvate kinase-M(I)-M(II)-ATP-Cr(III)-P-enolpyruvate complex, overlap of the transferred phosphoryl groups of the two substrates, which is required to explain the observed competition, is incomplete, allowing greater than or equal to 1 A for the transition state to form. In the active enzyme-M(I)-M(II)-ATP-Cr(III)-pyruvate complex, the gamma-phosphoryl phosphorus of ATP is in molecular contact (3.0 +/- 0.5 A) with the carbonyl oxygen of pyruvate, consistent with direct phosphoryl transfer, indicating no need for intermediate phosphorylation of the enzyme. The enzyme-bound divalent cation, which forms second sphere complexes with the phosphoryl groups of P-enolpyruvate and ATP, may activate the transferred phosphoryl group indirectly, through a water ligand. By analogy with the position of Cr(III), a second divalent cation may participate more directly by coordination of the triphosphate chain of ATP.