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 wildtype 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. (Blood. 2001;98:3113-3120)

Fifteen novel mutations inPKLRassociated with pyruvate kinase (PK) deficiency: Structural implications of amino acid substitutions in PK

“…The clinical history of this girl is described in more detail elsewhere [Raphaël et al, 2007]. This patient's DNA displayed two mutations: the splice-site mutation c.1269G4A, and c.1529G4A (p.Arg510Gln) of which the deleterious effects have been well documented [Kanno et al, 1997;Wang et al, 2001]. Surprisingly, a third and novel missense mutation was identified in cis with the c.1529G4A mutation.…”

“…It is the largest in-frame deletion thus far described in PKLR (www.pklrmutationdatabase.com) and it results in the deletion of six consecutive amino acids (ThrGlnGluLeuGlyThr) from the PK monomer (p.Thr48_Thr53). Deletion of these amino acids may affect the conversion of homotetrameric PK, as present in erythroblasts and young red blood cells, into the heterotetrameric form, which is present in mature red blood cells [Wang, et al, 2001]. Homotetrameric PK is composed of four 62-kDa subunits whereas the heterotetrameric form consists of two 62-kDa and two 57-58-kDa subunits [Kahn and Marie, 1982;Marie and Kahn, 1979].…”

“…Homotetrameric PK is composed of four 62-kDa subunits whereas the heterotetrameric form consists of two 62-kDa and two 57-58-kDa subunits [Kahn and Marie, 1982;Marie and Kahn, 1979]. In vitro studies on recombinant PK suggest that this conversion may involve the proteolytic removal of the first 47 amino acids of the 62-kDa PK monomer [Wang et al, 2001]. Hence, future studies may be aimed at investigating the effect of the naturally occurring p.Thr48_Thr53 mutant on proteolytic processing to gain more insight into the role of (removal of) the N-terminal part of PK-R in erythroid-specific regulation of PK.…”

Structure and Function of Human Erythrocyte Pyruvate Kinase

“…The induction time was 12 h, whereas the induction temperature was 30°C, with the exception of the mutants G332S, G364D, R504L, and R532W, for which the induction temperature was 21°C. Wild-type and mutant enzymes were purified by the procedure of Wang et al (13). Enzyme activities were measured at 37°C by the assay (13) recommended by the International Committee for Standardization in Hematology.…”

“…The crystals were difficult to reproduce. The recombinant enzyme undergoes partial proteolysis, and about 50% of the purified protein chains lack the first 47 amino acids (13). On this basis, we produced a mutant truncated en- e Analyzed with Procheck (38).…”