Conformational Differences in the Active Sites of Muscle and Erythrocyte Pyruvate Kinase

Abstract

Abstract We have examined the inhibition of the allosteric pyruvic kinase isozyme of human erythrocytes (PK I) and of the nonallosteric pyruvic kinase isozyme of rabbit muscle (PK III) by iodoacetamide and the protection against this inhibition by substrates and cations. Both isozymes are inhibited in a similar fashion which can be accounted for by alkylation of two different groups on each isozyme, one with a pKa of less than 6.8 and the other with a pKa of 7.8. Under the experimental conditions used, the effects observed in the protection studies were predominantly those involving the group with a pKa below 6.8. The rabbit muscle enzyme is not protected from iodoacetamide inhibition by phosphoenolpyruvate, ADP, or magnesium alone. It is protected by potassium or the magnesium-ADP complex. At pH 7.4, the human erythrocyte enzyme is not protected by phosphoenolpyruvate, ADP, or magnesium alone, or by potassium or the magnesium-ADP complex. In the presence of either of the positive allosteric effectors of the erythrocyte enzyme, phosphoenolpyruvate or fructose 1,6-diphosphate, both potassium and the magnesium-ADP complex have a protective action. We conclude that PK I exists in two conformational states, one equivalent to the T form of the Monod, Wyman, and Changeaux, model (Monod, J., Wyman, J., and Changeaux, G. P., J. Mol. Biol., 12, 88 (1965)) with a low affinity for phosphoenolpyruvate and no affinity for potassium or the magnesium-ADP complex, and the other equivalent to the R form of this model with a high affinity for phosphoenolpyruvate, potassium, and magnesium-ADP. Binding of either phosphoenolpyruvate or fructose 1,6-diphosphate converts the T form into the R form. PK III always exists in the R form with a high affinity for phosphoenolpyruvate, potassium, and the magnesium-ADP complex. We have demonstrated a cooperative influence of phosphoenolpyruvate on potassium binding to the human erythrocyte enzyme which is consistent with this allosteric model.