Enzymes of the red blood cell

Background

Red cell metabolism provides the cell with energy to pump ions against electrochemical gradients, maintain its shape, keep iron from hemoglobin in its reduced form, and maintain enzyme and hemoglobin sulfhydryl groups. The main source of metabolic energy comes from glucose. Glucose is metabolized through the Emden-Meyerhof glycolytic pathway and through the hexose monophosphate shunt, producing adenosine triphosphate (ATP) and nicotinamide-adenine dinucleotide (NADH). 2,3-Bisphosphoglycerate, an important regulator of the oxygen affinity of hemoglobin, is also generated during glycolysis. The hexose monophosphate shunt oxidizes glucose-6-phosphate, thereby generating nicotinamide adenine dinucleotide phosphate (NADPH). NADPH mainly serves the red cell to maintain high concentrations of reduced glutathione (GSH). The red cell lacks the capacity for de novo purine synthesis but has a salvage pathway that permits synthesis of purine nucleotides from purine bases.

Content

Hereditary red blood cell (RBC) enzymopathies are genetic disorders affecting genes encoding RBC enzymes involved in red cell metabolism. They cause a specific type of anemia designated hereditary nonspherocytic hemolytic anemia (HNSHA). HNSHA is a normocytic normochromic hemolytic anemia. In contrast to other hereditary red cell disorders, such as membrane disorders or hemoglobinopathies, morphologic abnormalities of the RBC are absent. The diagnosis is based on detection of reduced specific enzyme activity and molecular characterization of the defect on the DNA level. The most common enzyme disorders are deficiencies of glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase. However, there are a number of additional enzyme disorders, more rare and often much less known, causing HNSHA.

Hexokinase

Hexokinase (HK; Enzyme Classification [EC] number 2.7.1.1 [ https://www.qmul.ac.uk/sbcs/iubmb ]) catalyzes the phosphorylation of glucose to glucose-6-phosphate using ATP as a phosphoryl donor. The activity of HK is significantly higher in reticulocytes compared with mature red cells, where it is very low. The HK reaction is one of two rate-limiting steps in this pathway, the other being the phosphofructokinase reaction.

In mammalian tissues, four isozymes of HK with different enzymatic properties exist, HK-I to III with an Mr of 100 kDa, and HK-IV (or glucokinase) with an Mr of 50 kDa. HK-I is the predominant HK isozyme in tissues that depend strongly on glucose use for their physiologic functioning, such as brain, muscle, and red blood cells. HK-I displays unique regulatory properties in its sensitivity to inhibition by physiologic concentrations of the product G6P and relief of this inhibition by inorganic phosphate and by high concentrations of glucose. HK is a homodimer ^, and elucidation of the structures of human and rat HK-I has provided substantial insight into ligand-binding sites and subsequent modes of interaction. ^,

Apart from HK-I, RBCs contain a specific subtype of HK: HK-R. Both HKs are encoded by the gene HK1 , localized on chromosome 10q22 and spanning more than 100 kb. The structure of HK1 is complex: it encompasses 29 exons that, by tissue-specific transcription, generate multiple transcripts through alternative splicing of different 5′ exons. ^, Erythroid-specific transcriptional control results in a unique red cell-specific mRNA that differs from HK-I mRNA at the 5′untranslated region (5′-UTR) and at the first 63 nucleotides of the coding region. HK-R is mainly present in erythroblasts, reticulocytes, and young red cells. HK-1 replaces HK-R as the RBC matures and, as a result, mature red cells contain only 2 to 3% of the HK activity of reticulocytes.

HK deficiency (OMIM 235700; see Online Mendelian Inheritance in Man database: www.omim.org/ ) is a rare, recessively inherited disease with chronic nonspherocytic hemolytic anemia (CNSHA) as the predominant clinical feature. The phenotypic expression of the disease is heterogeneous, as with most glycolytic red cell enzyme deficiencies. The spectrum ranges from severe neonatal hemolysis and death to a fully compensated chronic hemolytic anemia. In general, patients benefit from splenectomy. Since HK activity is strongly dependent on red cell age, reticulocytosis, usually present in HK-deficient patients, may obscure the enzyme deficiency. Other age-dependent red cell enzymes (e.g., pyruvate kinase [PK], G6PD) should be measured simultaneously to assess the influence of reticulocyte enzyme activity.

Approximately 30 patients with HK deficiency have been described to date. Most patients are compound heterozygous for missense mutations in HK1 , some of which have been shown to affect enzyme stability ^, or the enzyme’s active site. A lethal case of HK deficiency was found to be due to a intragenic deletion of 9.5 kb, causing the deletion of exons 5 to 8 of HK1, resulting in a null allele. In three patients from two unrelated families, HK deficiency was found to be due to a mutation in the erythroid-specific promoter (−193A>G), which downregulates erythroid-specific transcription of HK1 and, hence, specifically affects HK-R production. These findings underscore the importance of including promoter mutation analysis in the molecular diagnosis of red cell enzyme deficiencies.

Mutations in HK1 have also been associated with Russe type hereditary motor and sensory neuropathy, retinitis pigmentosa 79, and neurodevelopmental abnormalities with visual impairment. In all these cases, HK enzymatic activity was normal and HNSHA was absent, suggesting a different pathogenic mechanism for the dominant missense variants in HK1 .

Phosphofructokinase

Phosphofructokinase (PFK; EC 2.7.1.11) catalyzes the irreversible phosphorylation of fructose-6-phosphate by ATP to fructose-1,6-bisphosphate (FBP; also called fructose 1,6-diphosphate, FDP). This conversion is rate limiting. PFK activity is tightly controlled by many metabolic effectors, ^, by binding to calmodulin, as well as the association of the enzyme with the red cell membrane.

The enzyme is a homotetramer or heterotetramer with a molecular mass of around 340 kDa. Three distinct isoenzymes have been identified in humans: PFK-M (muscle), PFK-L (liver), and PFK-P (platelet). In RBCs, the L- and M-isoforms are expressed; consequently, five forms of phosphofructokinase can be identified that differ in composition: M ₄ , M ₃ L ₁ , M ₂ L ₂ , ML ₃ , and L ₄ .

The genes encoding PFK-L ( PFKL ) and PFK-M ( PFKM ) are located on chromosome 21q22.3 and 12q13.3, respectively. The PFKM gene spans 30 kb, containing 27 exons and at least three promoter regions. PFKL contains 22 exons and spans more than 28 kb. A preliminary model of the structure of human muscle PFK has been presented.

PFK deficiency is a rare autosomal recessively inherited disorder. Because red cells contain both M and L subunits, mutations affecting either of the genes coding for these subunits will affect PFK activity. Thus when the L-subunit is affected, red cells contain only M ₄ PFK homotetramers and are partially PFK deficient. Similarly, when the M subunit is deficient, the partial PFK deficiency in red cells is accompanied by virtually absent PFK activity in muscle. This causes mild to severe myopathy, characterized by exercise intolerance, cramps, and myoglobinuria (Tarui disease or glycogen storage disease VII, OMIM 610681). The accompanying hemolysis is generally mild and may be absent. PFK-deficient red cells display a metabolic block at the PFK step in glycolysis and have decreased concentrations of 2,3-bisphosphoglyceric acid (2,3-BPG, also called 2,3-diphosphoglyceric acid, 2,3-DPG; see Rapoport-Luebering Shunt).

To date, approximately 100 cases with PFK deficiency have been reported and 25 different mutations in PFKM associated with PFK deficiency have been identified. About 60% of these mutations are missense mutations, with the remaining ones mainly affecting pre-mRNA processing. ^, Approximately one-third of identified PFK-deficient patients are of Ashkenazi Jewish origin. In this population, the most frequently encountered mutations are an intronic splice-site mutation in intron 5, c.237+1G>A, causing in-frame skipping of exon 5, and a single base-pair deletion in exon 22, c.2003delC, that disrupts the reading frame creating a premature stop codon (p.(Pro668Glnfs*17)).

To date, there has been only one reported case in which an unstable L subunit was identified. This patient exhibited no signs of myopathy or hemolysis.

The fact that PFK deficiency in dogs is associated with hemolytic crises after strenuous exercise, as well as the exercise intolerance, progressive cardiac hypertrophy, and reduced life span seen in Pfkm null mice indicate that Tarui disease is not simply a muscle glycogenosis, but rather a complex systemic disorder. ^,

PFK is relatively unstable, and PFK enzyme activity assays should be carried out on fresh blood samples, as activity will decrease rapidly upon prolonged storage.

Aldolase

Aldolase (fructose-bisphosphate aldolase; EC 4.1.2.13) catalyzes the reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. The enzyme is a 159-kDa tetramer of identical subunits of 40 kDa. Three isoenzymes have been identified to date: aldolases A, B, and C. Aldolase A is the isoenzyme that is expressed in the red cell but is also expressed in muscle. The flexible C-terminal region of aldolase A has been implicated in the catalytic function of the enzyme. ^, Red cell aldolase binds to actin, and the N-terminal domain of band 3, thereby inhibiting the enzyme’s activity. Aldolase activity is also influenced by red cell age.

The gene for aldolase A ( ALDOA ) is located on chromosome 16p11.2. It spans 7.5 kb and contains 12 exons. Several transcription-initiation sites were identified that direct tissue-specific splicing.

Aldolase deficiency (OMIM 611881) is a very rare disease; only seven cases have been described. All but one patient displayed chronic hemolytic anemia. In some patients, hemolysis is the sole clinical feature, whereas in other patients, hemolytic anemia is accompanied by myopathy, ^, rhabdomyolysis, psychomotor retardation, or mental retardation. ^, Intriguingly, the one patient without hemolytic anemia presented with severe fever-induced rhabdomyolysis, possibly due to tissue-specific thermal instability of the mutant aldolase.

Triosephosphate isomerase

Triosephosphate isomerase (TPI; EC 5.3.1.1) is the enzyme of the anaerobic glycolytic pathway with the highest activity. This ubiquitously expressed enzyme catalyzes the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. TPI is active as a dimer, consisting of two identical 27-kDa subunits of 248 amino acids. The three-dimensional structures of the human enzyme obtained by crystallography ^, show that the active site is located at the dimer interface and that several water molecules are an integral part of the dimer interface. No isoenzymes of TPI are known; only three distinct electrophoretic forms attributable to minor post-translational modifications have been identified. ^, RBC TPI activity is not related to red cell age.

TPI is transcribed from a single gene ( TPI1 ), located on chromosome 12p13. The gene spans 3.5 kb and contains seven exons that encode the 248 amino acids-long TPI subunit. Three processed pseudogenes have been found.

TPI deficiency (OMIM 190450) is a rare autosomal recessive systemic disorder and clinically the most severe disorder of glycolysis. The disease is characterized by hemolytic anemia, severe neuromuscular defects, increased susceptibility to infection, and cardiomyopathy. ^, Patients usually die in childhood, although intriguing exceptions have been reported. Because of the metabolic block at the TPI step, a 20- to 60-fold increase in red cell concentration of dihydroxyacetone phosphate occurs. The resulting elevated levels of toxic methylglyoxal, and consequent formation of advanced glycation end products is proposed to be a key factor in the pathophysiology of TPI deficiency-related severe neuromuscular disease. In addition, molecular changes in the TPI dimer interface may cause a functional synaptic defect in TPI deficiency resulting in neurologic dysfunction.

To date, 20 different mutations, mostly missense, have been identified in the gene encoding TPI, and approximately 50 patients have been reported. Mutations result in decreased enzymatic activity and/or dissociation of the TPI dimer into inactive monomers. The most common mutation is c.315G>C p.(Glu105Asp), a mutation detected in approximately 80% of patients. While TPI activity itself is not affected, this mutation impairs the formation of active dimers by disrupting the water-protein and water-water interactions that join the two monomers. Haplotype analysis suggest a single origin for this mutation with the common ancestor in Northern Europe.

Phosphoglycerate kinase

Phosphoglycerate kinase (PGK, EC 2.7.2.3) catalyzes the reversible conversion of 1,3-bisphosphoglycerate (also called 1,2-diphosphoglycerate) to 3-phosphoglycerate, thereby generating one molecule of ATP. The reaction can be bypassed by the Rapoport-Luebering shunt at the expense of one molecule of ATP (see Rapoport-Luebering shunt and Fig. 78.1 ) This alternative routing of glycolytic intermediates has been called the energy clutch of glycolysis.

In humans, two isoenzymes (PGK-1 and PGK-2) exist. PGK-1 is ubiquitously expressed in all somatic cells; PGK-2 is expressed only in spermatozoa. PGK-1 is a 48-kDa monomeric enzyme consisting of 417 amino acids. The gene encoding PGK-1 ( PGK1 ) is located on the long arm of the X-chromosome (Xq13). The gene spans 23 kb and is composed of 11 exons. Nonfunctional pseudogenes have been found on the X chromosome and chromosome 19.

PGK deficiency (OMIM 311800) is one of the uncommon causes of HNSHA. Sometimes the deficiency manifests only mild to severe chronic hemolytic anemia, but in many other cases other clinical findings are present, particularly neurologic symptoms and myopathy; these may occur with or without hemolytic anemia. More than 50 patients with PGK deficiency have been characterized. Multiorgan involvement in patients with PGK deficiency appears to be associated with lower residual enzyme activity, as compared to patients displaying only myopathy. Most of the 29 mutations reported to date are missense ones, mainly affecting thermal stability of the protein. They are mostly unique mutations except for the c.491A>T (p.Asp164Val) change, which has been encountered three times, each in the context of a different haplotype, suggesting independent origins of this mutation. Review of amino acid substitutions in PGK has gained some insight into the genotype-to-phenotype correlation in PGK deficiency, but the reason for the range of manifestations in PGK deficiency remains unclear, suggesting that other yet unknown environmental, metabolic, genetic, and/or epigenetic factors are involved. ^,

Pyruvate kinase

Pyruvate kinase (PK; EC 2.7.1.40) catalyzes the conversion of phosphoenolpyruvate to pyruvate with the concomitant generation of the second molecule of ATP in glycolysis (see Fig. 78.1 ). Pyruvate is crucial for several metabolic pathways, and PK represents one of the major regulatory enzymes of glycolysis. PK is allosterically activated by its substrate and by FBP (fructose-1,6-bisphosphate), and its enzymatic activity strongly depends on red cell age. Therefore because the youngest red cells have the highest activity, a deficiency of PK may easily be masked by reticulocytosis.

PK is a homotetrameric enzyme. In mammals, four isozymes are expressed. PK-M1 is expressed in skeletal muscle, heart, and brain. It is the only PK isozyme that is not allosterically regulated. PK-M2 is expressed in early fetal tissues and in adult tissues, including leukocytes and platelets. Both M1 and M2 isozymes are produced from a single gene ( PKM2 ) by means of alternative splicing. ^, PK-L is predominantly expressed in the liver, whereas the expression of PK-R is confined to the red cell. The PK-L and PK-R subunits are transcribed from a single gene ( PKLR ) located on chromosome 1q21 by the use of tissue-specific promoters. ^, The PKLR gene consists of 12 exons and is approximately 9.5 kb in size. Exon 1 is exclusively expressed in erythroid cells, whereas expression of exon 2 is confined to the liver. Hence, the PK-R monomer is composed of 574 amino acids. The PK-L subunit comprises 531 amino acids.

In basophilic erythroblasts, both the PK-M2 and PK-R isozymes are expressed. During further erythroid differentiation and maturation the PK-R isozyme progressively replaces PK-M2. In addition, the red cell limited proteolytic degradation of the 63-kDa PK-R subunit renders a subset of PK-R monomers of 57 to 58 kDa. Consequently, in young and mature human red cells two distinct species can be distinguished, PK-R1 and PK-R2, that differ in PK-R and “processed” PK-R subunit composition.

The crystal structure of human red cell PK has been elucidated. Each PK-R subunit is composed of four domains: N, A, B, and C. The active site lies in a cleft between the A-domain and the flexible B-domain. The B-domain is capable of rotating with respect to the A-domain, generating either the “open” or “closed” conformation. The C-domain contains the binding site for FBP. In the PK tetramer, subunit interactions at the interfaces between the A and C domains, as well as A/B and A/C domain interactions, within one subunit are considered to be key determinants of the allosteric response, involving switching from the low-affinity T-state to the high affinity R-state.

Pyruvate kinase deficiency (OMIM 266200) is the most common cause of nonspherocytic hemolytic anemia due to defective glycolysis. It is an autosomal recessive disease. In the general white population, the allelic frequency is estimated to be around 2%.

The two major metabolic abnormalities resulting from PK deficiency are ATP depletion and increased levels of 2,3-BPG. The precise mechanisms by which the enzyme deficiency leads to a shortened RBC lifespan are unknown. An important feature, however, is the selective sequestration of PK-deficient reticulocytes by the spleen. It has been suggested that the metabolic disturbances of the enzyme deficiency may affect not only red cell survival, but also the maturation of PK-deficient erythroid progenitors, resulting in ineffective erythropoiesis.

PK-deficient patients display a highly variable degree of chronic hemolysis with variable clinical severity. Clinical symptoms range from severe anemia and death at birth, severe transfusion-dependent chronic hemolysis, or moderate hemolysis with exacerbation during infection, to a well-compensated hemolysis without anemia. Common complications are iron overload and gallstones, and perinatal complications include anemia requiring transfusion, hyperbilirubinemia, hydrops, and prematurity. Splenectomy is, in general, beneficial, as it is associated with an increase in hemoglobin and decreased transfusion burden. PK deficiency has been treated successfully by stem cell transplantation. Gene therapy strategies have been shown to be able to correct the PK-deficient phenotype in mice. Recent evidence indicates that small molecule activation of mutant PK restores glycolysis and normalizes red cell metabolism in PK deficiency, and clinical trials using small molecule allosteric activators of PK have reported an increase in hemoglobin level in half of the PK-deficient patients who were administered the drug.

To date, more than 260 mutations in PKLR have been reported to be associated with pyruvate kinase deficiency. Two-thirds of these mutations are missense mutations affecting conserved residues in structurally and functionally important domains of PK. The most frequently detected mutations are missense mutants c.1456C>T (p.Arg486Trp), c.1529G>A (p.Arg510Gln), c.994G>A (p.Gly332Ser), and nonsense mutant c.721G>T (p.Glu241*). Evaluating the protein structural context of affected residues using the three-dimensional structure of recombinant human tetrameric PK has provided a rationale for the observed enzyme deficiency. ^{, ,} From a large cohort of PK-deficient patients, a limited genotype-to-phenotype correlation was identified: patients with two missense mutations had a lower likelihood of splenectomy, fewer transfusions, and a lower rate of iron overload, whereas patients with two non-missense mutations were less likely to have a complete or partial response to splenectomy. It is important to note that because most PK-deficient patients are compound heterozygous for two different (missense) mutations, up to seven different tetrameric forms of PK may be present in such patients, each with distinct structural and kinetic properties. This complicates genotype-to-phenotype correlations as it is difficult to infer which mutation is primarily responsible for deficient enzyme function and the clinical phenotype.

Pyruvate kinase deficiency has been reported to have a protective effect against replication of the malarial parasite in human RBCs, ^, and malaria has been found to act as a selective force in the PKLR genomic region. This protective effect may be related to the reduced ATP levels in PK-deficient red blood cells.

PK levels are also decreased in patients with red cell disorders caused by mutations in KLF1, probably due to altered binding of mutant KLF1 to the erythroid promoter region of PKLR .

Lactate dehydrogenase

LD catalyzes the conversion of pyruvate to lactate, the last step in the EMP. Deficiency of this enzyme is not associated with hematologic disease. LD is described in Chapter 32 .