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Blood, 15 November 2001, Vol. 98, No. 10, pp. 3113-3120
RED CELLS
Human erythrocyte pyruvate kinase: characterization of the
recombinant enzyme and a mutant form (R510Q) causing nonspherocytic
hemolytic anemia
Changqing Wang,
Laurent R. Chiarelli,
Paola Bianchi,
Donald J. Abraham,
Alessandro Galizzi,
Andrea Mattevi,
Alberto Zanella, and
Giovanna Valentini
From the Department of Biochemistry, Department of
Genetics and Microbiology, University of Pavia, Pavia, Italy;
Department of Medicinal Chemistry, Virginia Commonwealth University,
Richmond, VA; and Hematology Division, I.R.C.C.S. Ospedale Maggiore,
Milano, Italy.
Human erythrocyte pyruvate kinase plays an important role in
erythrocyte metabolism. 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 mutations 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 purified. The
tetrameric protein exhibited properties characteristic of authentic
human erythrocyte pyruvate kinase, including response to substrate,
phosphoenolpyruvate, 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 cleaved and lacked 47 N-terminal amino acid residues. A
mutant protein, R510Q, which is the most frequently occurring mutation
among Northern European population, was also generated and purified.
The mutant protein retained its binding capacity to and could be
activated by fructose 1,6-bisphosphate and showed similar kinetics
toward phosphoenolpyruvate and adenosine diphosphate as for the
wild-type enzyme. Conversely, the mutant protein has a dramatically
decreased stability toward heat and is more susceptible to ATP
inhibition. The enzyme instability decreases the enzyme level in the
cell, accounting for the clinically observed "pyruvate kinase
deficiency" of patients who are homozygous for this mutation. This
study provides the first detailed functional characterization of human
erythrocyte pyruvate kinase. These findings will allow the
establishment of a fine correlation between molecular abnormalities and
the clinical expression of the disease.
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