Abstract
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).
Highlights
Adenine nucleotides were found to promote inactivation by MalNEt and phosphoenolpyruvate protected against MalNEt
We present evidence that this lag phase results from a slow, P-enolpyruvate-mediated conversion of the enzyme to a more active form
To facilitate study of this lag phase, the extent was estimated by taking the ratio (R,) of the slope of the initial, slow phase of the progress curve to the slope of the final, fast phase
Summary
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. Pyruvate kinase (EC 2.7.1.40, ATP-pyruvate phosphotransferase) occupies a central role in the metabolism of the human erythrocyte. It catalyzes one of the two ATP-producing reactions in this cell type and is important in establishing the steady state levels of 2,3-Pz-glycerate’ (l-3). We present evidence that this lag phase results from a slow, P-enolpyruvate-mediated conversion of the enzyme to a more active form. In contrast, were found to promote formation of the less active form
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