Human erythrocyte pyruvate kinase: characterization of the recombinant enzyme and a mutant form (R510Q) causing nonspherocytic hemolytic anemia

Human erythrocyte pyruvate kinase plays an important role in erythrocyte me tabolism. Mutation on the gene results in pyruvate kinase deficiency and is an important cause of hereditary nonspherocytic hemolytic anemia. Because of difficulties in isolating the mutant enzymes from patients, these mutati ons have not been fully studied. In this study, a complementary DNA (cDNA) encoding the human erythrocyte pyruvate kinase was generated. The cDNA was cloned into several expression vectors, and the protein was expressed and p urified. The tetrameric protein exhibited properties characteristic of auth entic human erythrocyte pyruvate kinase, including response to substrate, p hosphoenolpyruvate, activation by fructose 1,6-bisphosphate, and inhibition by adenosine triphosphate (ATP). The N-terminal segment of the protein was highly susceptible to proteolysis, but only 2 of the 4 subunits were cleav ed and lacked 47 N-terminal amino acid residues. A mutant protein, R510Q, w hich is the most frequently occurring mutation among Northern European popu lation, was also generated and purified. The mutant protein retained its bi nding capacity to and could be activated by fructose 1,6-bisphosphate and s howed similar kinetics toward phosphoenolpyruvate and adenosine diphosphate as for the wildtype enzyme. Conversely, the mutant protein has a dramatica lly decreased stability toward heat and is more susceptible to ATP inhibiti on. The enzyme instability decreases the enzyme level in the cell, accounti ng for the clinically observed "pyruvate kinase deficiency" of patients who are homozygous for this mutation. This study provides the first detailed f unctional characterization of human erythrocyte pyruvate kinase. These find ings will allow the establishment of a line correlation between molecular a bnormalities and the clinical expression of the disease. (C) 2001 by The Am erican Society of Hematology.