Structural changes at the active site of pyruvate kinase during activation and catalysis.

The distance between the obligatory monovalent and divalent cations at the active site of pyruvate kinase (rabbit muscle) has been used to monitor structural changes at various successive intermediates in the catalytic reaction and to relate structure and activity of substrate analogs. Determinations of distance were obtained from measurements of the longitudinal relaxation rate (1/T1) of the methyl protons of methyl ammonium ion, which was affected by the activating divalent cation Mn2+ in the various enzyme complexes. A frequency dependence of the 1/T1 effects with several complexes indicats that the observed relaxation rate changes are modulated by changes in the cation-cation distances. The results are interpreted as a sequence of structural changes at the active site which occur upon substrate binding, catalysis, and product release. Substrate analogs which are good analogs of phosphoenolpyruvate by kinetic criteria induce structural changes analogous to the substrate. An attempt is made to correlate the cation-cation distance changes to other structural changes reported for pyruvate kinase.