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894 Part VI: The Erythrocyte Chapter 58: The Porphyrias 895

rearrangement that affects only ring D of the porphyrin macrocycle (see 354 amino acid residues (Mr = 40,647 Da), with a putative leader sequence
Fig. 58–4, step 4). In the absence of this enzyme, HMB spontaneously of 31 amino acid residues. The result is a mature protein consisting of 323
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forms the ring structure uroporphyrinogen I, which, like the III isomer, amino acid residues (Mr = 37,225 Da). Potential regulatory elements
is a substrate for uroporphyrinogen decarboxylase (UROD). However, exist in the GC-rich promoter region of the gene, such as six Sp1, four
because coproporphyrinogen I is not a substrate for coproporphyrinogen GATA-1, one CACCC site, and the CPO gene promoter regulatory element
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oxidase (CPO) the type I porphyrinogen isomers are not further metabo- (CPRE). CPRE binds specifically to a CPRE-binding protein, which has a
lized, and only the type III isomers are precursors of heme. leucine-zipper-like structure and serves as a DNA sequence-specific
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The UROS cDNA has an open-reading frame of 798 bp, and the transcription factor that regulates gene expression. Tissue-specific
predicted protein product consists of 263 amino acid residues, with an expression of CPO is significant. For example, binding proteins to
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Mr of 28,607 Da. The amino acid compositions of the hepatic and the the Spl-like element, CPRE and GATA-1, cooperatively function in
purified erythrocyte enzyme are essentially identical, and no tissue- CPO gene expression in erythroid cells. The CPRE-binding protein by
specific isoforms have been described. itself plays a principal role in basal expression of CPO in nonerythroid
The interspecies homology for the UROS proteins is below 10 cells. CPO mRNA increases during erythroid cell differentiation.
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percent, depending on the number and divergence of the species being Newly synthesized human CPO contains a 110-amino-acid N-terminal
compared. However, the crystal structures of uroporphyrinogen III syn- signal peptide, which is removed during transport into the intermem-
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thase from human and Thermus thermophilus have been solved and are brane space of mitochondria, yielding a mature protein of 354 amino acid
very similar. 60,61 The structure supports a mechanism that includes the residues (Mr = 36,842 Da). A five-base insertional mutation in the middle
formation of a spirolactam intermediate by positioning the A and D of this presequence has been described in one patient with HCP. 75
rings such that the noncatalytic closure, to form uroporphyrinogen I, Protoporphyrinogen Oxidase (EC 1.3.3.4) The penultimate step
is not possible. 61 in heme biosynthesis is the oxidation of protoporphyrinogen IX to pro-
Uroporphyrinogen Decarboxylase (EC 4.1.1.37) UROD is a toporphyrin IX, with removal of six hydrogen atoms. This reaction is
cytosolic enzyme that catalyzes the sequential removal of the four car- mediated by the mitochondrial enzyme protoporphyrinogen oxidase
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boxylic groups of the carboxymethyl side chains in uroporphyrinogen (PPO; see Fig. 58–4, step 7). Human PPO cDNA has been cloned.
to yield coproporphyrinogen (see Fig. 58–4, step 5). The four successive The gene is present as a single copy per haploid genome, at chromo-
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decarboxylation reactions yield 7-, 6-, 5-, and 4-carboxylated porphy- some 1q22. PPO consists of 477 amino acids with an Mr of 50,800 Da.
rinogens. Increased amounts of these intermediates can be identified The deduced protein exhibits a high degree of homology over its entire
as the corresponding oxidized porphyrins in liver, plasma, urine and length to the amino acid sequence of PPO encoded by the HEMY gene
stool in human PCT and in laboratory animal models in which hepatic of Bacillus subtilis. PPO has been crystallized and the structure shows
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UROD is inhibited. An inhibitor of UROD activity, a partially oxidized that the enzyme is a homodimer. Sequences required for import into
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substrate molecule, is produced in liver of experimental animals in the mitochondria have been identified. Expression of PPO is upreg-
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response to halogenated polycyclic aromatic hydrocarbons such as hex- ulated, approximately fourfold, in the developing erythron from two
achlorobenzene, dioxin, and polychlorinated biphenyls, as well as other GATA-1 binding sites located in exon 1. 80
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compounds able to activate the Ah receptor. This porphomethene Ferrochelatase (Protoheme-Ferrolyase; EC 4.99.1.1) The final
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compound is believed to explain UROD inhibition in human PCT. step of heme biosynthesis is the insertion of iron into protoporphyrin
Human UROD is a 42-kDa polypeptide encoded by a single gene con- IX. This reaction is catalyzed by the mitochondrial enzyme ferrochela-
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taining 10 exons spread over 3 kb and functions as a homodimer. The tase (FECH; see Fig. 58–4, step 8). The enzyme uses protoporphyrin IX,
gene has been mapped to chromosome 1p34. 65 rather than its reduced form, as substrate, but requires the reduced fer-
Although the UROD gene contains two initiation sites, both sites rous form of iron. The gene encoding human FECH has been assigned
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are used with the same frequencies in all tissues, and the gene is tran- to chromosome 18q. Two FECH mRNA species, approximately 2.5 kb
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scribed into a single mRNA. Recombinant human UROD purified to and approximately 1.6 kb in size, are derived from the utilization of two
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homogeneity has been crystallized, and its crystal structure was deter- alternative polyadenylation sites in the mRNA. The human FECH gene
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mined at 1.60-Å resolution. The purified protein is a dimer with a contains a total of 11 exons and has a minimum size of approximately
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dissociation constant of 0.1 μM. The 40.8-kDa polypeptide forms a 45 kb. A major site of transcription initiation is at an adenine, 89 bp
single domain with a distorted (β/α) -barrel fold, and a distinctive deep upstream from the translation-initiating ATG. The promoter region
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cleft for the enzyme’s active site is formed by loops at the C-terminal contains a potential binding site for several transcription factors, Sp1,
ends of the barrel strands. The protein forms a homodimer with one NF-E2, and GATA-1, but not a typical TATA or CAAT sequence. The
active-site cleft per monomer located adjacent to its neighbor in the transcripts are identical in all tissues examined.
dimer. The structure creates a single extended cleft that is large enough The crystal structure of B. subtilis FECH has been determined at
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to accommodate two substrate molecules in close proximity. Although 1.9-Å resolution. Subsequently the structure of human FECH was
both uroporphyrinogen I and III are metabolized by UROD, only the solved and the location of the substrate binding site determined. The
coproporphyrinogen III isomer is further metabolized to heme. 68 enzyme functions as a homodimer and associates with the inside of the
Coproporphyrinogen Oxidase (EC 4.1.1.37) CPO is located on inner mitochondrial membrane. The mechanism of catalysis has not
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the outer surface of the inner mitochondrial membrane in mammalian been identified nor has a function been assigned to the Fe- S cluster that
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cells. The enzyme catalyzes the removal of the carboxyl group and two is present in human FECH. Lead inhibits FECH, and a structure of the
hydrogens from the propionic groups of pyrrole rings A and B, forming protein-lead complex has been solved indicating a critical role for the
vinyl groups at these positions (see Fig. 58–4, step 6). The enzyme is iso- pi helix in catalysis. FECH seems to have a structurally conserved core
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mer specific for coproporphyrinogen III, yielding protoporphyrinogen region that is common to the enzyme from bacteria, plants, and mammals.
IX (see Fig. 58–4, step 6). The gene for human CPO has been assigned
to chromosome 3q12, spans approximately 14 kb, and consists of seven Control of Heme Synthesis in the Liver and Erythroid Cells
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exons and six introns. cDNA cloning for this enzyme was first reported Tissue-specific aspects of heme synthesis have been studied mostly
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in mouse erythroleukemia cells. The predicted mouse protein comprises in erythroid cells and hepatocytes, as the marrow and liver have the
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