title: Human erythrocyte pyruvate kinase: characterization of the recombinant enzyme and a mutant form (R510Q) causing nonspherocytic hemolytic anemia.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Pavia, Pavia, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Wang
authors.forename: L R
authors.initials: LR
authors.lastname: Chiarelli
authors.forename: P
authors.initials: P
authors.lastname: Bianchi
authors.forename: D J
authors.initials: DJ
authors.lastname: Abraham
authors.forename: A
authors.initials: A
authors.lastname: Galizzi
authors.forename: A
authors.initials: A
authors.lastname: Mattevi
authors.forename: A
authors.initials: A
authors.lastname: Zanella
authors.forename: G
authors.initials: G
authors.lastname: Valentini
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000746:Anemia, Hemolytic, Congenital Nonspherocytic / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004912:Erythrocytes / Q000201:enzymology*; D005635:Fructosediphosphates / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000172:deficiency / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020125:Mutation, Missense; D010728:Phosphoenolpyruvate / Q000378:metabolism; D017354:Point Mutation; D011489:Protein Denaturation; D021122:Protein Subunits; D011770:Pyruvate Kinase / Q000737:chemistry / Q000172:deficiency / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D018076:DNA, Complementary; D005635:Fructosediphosphates; D007527:Isoenzymes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; C029063:fructose-1,6-diphosphate