Mechanisms of fructose diphosphate activation of a mutant pyruvate kinase from human red cells

Abstract

1. 1. Normal human erythrocyte pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) and mutant pyruvate kinase from a propositus with congenital, nonspherocytic, hemolytic anemia were isolated by DEAE-cellulose extraction and (NH 4) 2SO 4 precipitation. 2. 2. Fru-1,6-P 2 lowered the apparent Michaelis constant for phosphoenolpyruvate of the normal enzyme but did not substantially change the maximal velocity. This activator greatly elevated both the apparent Michaelis constant for phosphoenolpyruvate and the maximalvelocity of the mutant enzyme. 3. 3. Fru-1,6-P 2 did not affect the Michaelis constant for ADP or the optimal Mg 2+ concentration in either the normal or the mutant enzyme. 4. 4. Fru-1,6-P 2 altered the extremely sensitive interaction of the binding sites for the two substrates on the mutant enzyme. 5. 5. The sensitivity of the normal enzyme to Fru-1,6-P 2 could be increased by either dilution or heating. With heating, dilute solutions of both normal and mutant enzymes decayed in two steps, indicating the presence of multiple conformations in both enzymes. 6. 6. A molecular model is proposed for Fru-1,6-P 2 activation; Fru-1,6-P 2 acts directly on the phosphoenolpyruvate binding site; interaction of the two substrate sites then alters the conformation of the binding site for ADP.