Evidence of a dissociative SN1(P) mechanism of phosphoryl transfer by rabbit muscle pyruvate kinase

Abstract

Adenosine 5′-[αβ-¹⁸O,β-¹⁸O₂]triphosphate has been prepared and used to investigate the mechanism of action of rabbit muscle pyruvate kinase. In the presence of pyruvate kinase and pyruvate, randomisation of the β-¹⁸O₂ labels with the βγ-O bridge occurs as expected. However randomisation also occurs in the presence of the potent inhibitor oxalate, an analogue of enol pyruvate, and also in the absence of pyruvate or a pyruvate analogue. Since there is evidence against the involvement of a phosphoenzyme intermediate in the reaction pathway of pyruvate kinase, the results indicate that phosphoryl transfer in pyruvate kinase occurs by a dissociative S_N1(P) mechanism, involving the transient high energy but tightly enzyme bound metaphosphate ion intermediate. The rate constants for the pyruvate kinase catalysed ¹⁸O-redistribution in adenosine 5′-[αβ-¹⁸O,β-¹⁸O₂]triphosphate alone and in the presence of oxalate and pyruvate are 1.1 × 10² min^–1, 2.0 × 10² min^–1, and 2.0 × 10³ min^–1 respectively.