Hysteretic response of human erythrocyte pyruvate kinase to phosphoenolpyruvate. Potential role in regulation of 2,3-bisphosphoglycerate metabolism.

Human erythrocyte pyruvate kinase (EC 2.7.1.40, ATP-pyruvate phosphotransferase) was found to display a time-dependent activation (lag phase) in the reaction progress curves. The extent of this lag phase depended upon the treatment of the enzyme prior to assay. Preincubation of the enzyme with adenine nucleotides amplified the lag, whereas prior treatment with phosphoenolpyruvate diminished it. The activation process was first order in enzyme with the pseudo first order rate constants being a hyperbolic function of phosphoenolpyruvate concentration. The data provide evidence for a phosphoenol-pyruvate-mediated conversion of the enzyme to a more active form. Studies with the irreversible sulfhydryl inhibitor, N-ethylmaleimide (MalNEt), provided additional evidence for different conformational states of the enzyme induced by its substrates and effectors. Adenine nucleotides were found to promote inactivation by MalNEt and phosphoenolpyruvate protected against MalNEt. The possible metabolic significance of this "hysteretic" pyruvate kinase is discussed in relation to the known role of this enzyme in 2,3-bisphosphoglycerate metabolism (Rose, I.A. (1971) Exp. Eye Res. 11, 264-272).