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694 Part VI: The Erythrocyte Chapter 47: Erythrocyte Enzyme Disorders 695

for activity. PFK activity is tightly controlled and subject to regulation reveals a homotetramer, each subunit of which is bound to a NAD
+
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by many metabolic effectors. Among the most important activators molecule. Deficiency of GAPDH seems a rare occurrence without
are ADP, cyclic adenosine monophosphate (cAMP), and fructose-2,6- functional consequences. 94
diphosphate, whereas PFK is inhibited by its substrate ATP, citrate, and Phosphoglycerate Kinase PGK effects the transfer to ADP of the
lactate. 64,65 These metabolic effectors exert their effects probably by sta- high-energy phosphate from the 1-carbon of 1,3- diphosphoglycerate
bilizing either the minimally active dimeric and fully active tetrameric (1,3-DPG) to form ATP. The reaction is readily reversible and can be
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form of PFK. PFK activity is also regulated by its binding to calm- bypassed by the Rapoport-Luebering shunt. The isoenzyme PGK-1
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odulin, and association of the enzyme with the red cell membrane ; is ubiquitously expressed in all somatic cells and is a 48-kDa mono-
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in particular, binding to band 3 69,70 and actin appears to inhibit and meric enzyme of 417 amino acids. Expression of isozyme PGK-2
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stimulate PFK activity, respectively. has been found in testis. PGK is composed of two domains. The N-
A preliminary crystallographic analysis of dimeric wild-type terminal domain binds 3-phosphoglycerate and 1,3-DPG, whereas
human muscle PFK has been presented. The gene encoding the 85-kDa ADP and ATP bind to the C-terminal domain. For catalysis to occur,
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M subunit (PFKM) is located on chromosome 12q13.3 and spans 30 kb. the protein needs to undergo a large conformational change (“hinge
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It contains 27 exons and at least three promoter regions. The 80-kDa bending”). 97–99 The gene encoding PGK (PGK-1) is located on the long
L-subunit encoding gene (PFKL) is located on chromosome 21q22.3; arm of the X-chromosome (Xq13), spans 23 kb, and is composed of 11
it contains 22 exons and spans more than 28 kb. Deficiency of PFK exons. Nonfunctional pseudogenes have been located on chromosome
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is associated with mild hemolytic anemia and with type VII glycogen 19 and the X-chromosome. Deficiency of PGK is a rare cause of non-
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storage disease (Tarui disease). 75 spherocytic hemolytic anemia, often associated with neuromuscular
Aldolase Aldolase reversibly cleaves fructose-1,6-diphosphate into abnormalities.
two trioses. The “upper” half of the fructose-1,6-diphosphate molecule Bisphosphoglycerate Mutase The same protein molecule is
becomes dihydroxyacetone phosphate and the “lower” half becomes responsible for both bisphosphoglycerate mutase and bisphosphoglyc-
glyceraldehyde-3-phosphate. Aldolase is a 159-kDa homotetrameric erate phosphatase activities in the erythrocyte. 35,100 This enzyme is par-
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enzyme comprised of subunits of 40 kDa each. Three distinct isoen- ticularly important because it regulates the concentration of 2,3-BPG
zymes have been identified: aldolase A, B, and C. The 364-amino-acids- of erythrocytes. In its role as a bisphosphoglyceromutase, the enzyme
long aldolase A subunits are primarily expressed in erythrocytes and competes with PGK for 1,3-BPG as a substrate. It changes 1,3-BPG to
muscle cells. The structure of human aldolase A is known. Red cell 2,3-BPG, thereby dissipating the energy of the high-energy acylphos-
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aldolase binds to F-actin and the N-terminal part of band 3, which phate bond. It is inhibited by its product 2,3-BPG and by inorganic
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inhibits its activity. The gene for aldolase A (ALDOA) is located on phosphate, and it is activated by 2-phosphoglycerate and by increased
chromosome 16q22–24. It spans 7.5 kb and consists of 12 exons. Several pH levels. It requires 3-phosphoglycerate for activity. In its role as bis-
transcription-initiation sites were identified and ALDOA pre-mRNA is phosphoglycerate phosphatase it catalyzes the removal of the phosphate
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spliced in a tissue-specific manner. Aldolase deficiency is a very rare group from carbon 2 of 2,3-BPG. It is inhibited by its product 3-
cause of hereditary nonspherocytic hemolytic anemia. phosphoglycerate and by sulfhydryl reagents. It is most active at a
Triosephosphate Isomerase TPI is the enzyme of the anaerobic slightly acid pH and is strongly stimulated by bisulfite and phospho-
glycolytic pathway that has the highest activity. Its metabolic role is to glycolate. Phosphoglycolate, the most potent activator of phosphatase
catalyze interconversion of the two trioses formed by the action of aldo- activity, is present in erythrocytes at very low concentrations, but the
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lase: dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. source of this substance in red cells is unknown. 103,104 Phosphoglycolate
Although equilibrium is in favor of dihydroxyacetone phosphate, phosphatase, the enzyme that hydrolyzes phosphoglycolate, has also
glyceraldehyde-3-phosphate undergoes continued oxidation through been identified in erythrocytes. 105
the action of GAPDH and is thus removed from the equilibrium. TPI Bisphosphoglycerate mutase is a homodimer, with 30-kDa subun-
is a dimer consisting of two identical 27-kDa subunits of 248 amino its consisting of 258 amino acids. The crystal structure of human bis-
acids. Several crystal structures have been resolved. 84,85 These show phosphoglycerate mutase has been determined, providing a rationale
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that the active site is at the dimer interface and a number of critical res- for the specific residues that are crucial for synthase, mutase, and phos-
idues have been identified. Several water molecules, some of which are phatase activity. 106,107 The gene for bisphosphoglycerate mutase (BPGM)
highly conserved, are an integral part of the dimer interface. There are has been mapped to chromosome 7q31–34 and it consists of 3 exons,
no isoenzymes known, but three distinct electrophoretic forms can be spanning more than 22 kb.
distinguished as a result of posttranslational modifications. Red blood A deficiency of bisphosphoglycerate mutase results in a marked
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cell TPI activity is not red-cell-age dependent. TPI is transcribed from decrease in red cell 2,3-BPG levels. The consequent left shift of the oxy-
a single gene (TPI1), located on chromosome 12p13. The gene spans 3.5 gen dissociation curve leads to erythrocytosis/polycythemia (Chap. 57).
kb and contains 7 exons. Three processed pseudogenes have been iden- Phosphoglycerate Mutase An equilibrium is established between
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tified. A deficiency of TPI causes hereditary nonspherocytic hemolytic 3-phosphoglycerate and 2-phosphoglycerate by phosphoglycerate
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anemia and a severe neuromuscular disorder. mutase. 2,3-BPG acts as an essential cofactor for the transforma-
Glyceraldehyde Phosphate Dehydrogenase GAPDH performs tion. Red blood cell phosphoglycerate mutase is a heterodimer con-
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the dual functions of oxidizing and phosphorylating glyceraldehyde-3- sisting of M and B subunits, encoded by separate genes. Only one
phosphate, producing 1,3-BPG. In the process, NAD is reduced to well-characterized patient with partial red blood cell monophospho-
+
NADH. This enzyme is closely associated with the red cell membrane glycerate mutase deficiency has been described who was homozygous
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where it binds to the N-terminal part of band 3. Membrane binding for a p.Met230Ile amino acid change in the B subunit. The mutant
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influences the activity of GAPDH thereby possibly regulating gly- enzyme was unstable and thus had a short half-life. Unexpectedly,
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colytic flux. Human red blood cell GAPDH has been purified. It is a all glycolytic intermediates were decreased, possibly because of lactate
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homotetramer of approximately 150 kDa, composed of 36-kDa sub- accumulation. The exact clinical consequences of this red blood cell
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units, and shows an absolute specificity for NAD . One of the many enzymopathy remain to be established.
nonglycolytic functions of GAPDH 40,41 may include its function as a Enolase Enolase is a homodimeric enzyme that establishes an
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transferrin receptor. The crystal structure of human liver GAPDH equilibrium between 2-phosphoglycerate and phosphoenolpyruvate.
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