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

in liver, accounting for the beneficial effects of intravenous treatment N-terminal arm. Pairs of monomers then wrap their arms around each
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of the acute porphyrias with hemin. At higher concentrations, heme other to form compact dimers, and these dimers associate to form a
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induces heme oxygenase, resulting in its enhanced catabolism. Thus, 422 symmetric octamer. All eight active sites are on the surface of the
hepatic heme availability is balanced between the rate of synthesis octamer and possess two lysine residues (210 and 263). The Lys263 res-
controlled primarily by ALAS1 and the rate of degradation controlled idue forms a Schiff base link to the substrate. The two lysine side chains
by heme oxygenase, both of which are regulated by heme at different are close to two zinc binding sites. One binding site is formed by three
intracellular concentrations. ALAS1 is also upregulated by the perox- cysteine residues; the other involves Cys234 and His142.
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isomal proliferator-activated cofactor 1α (PGC-1α), a coactivator of Although there are no tissue-specific ALAD isozymes, the ALAD
nuclear receptors and transcription factors. Transcriptional regulation mRNA has two splice variants, a housekeeping (1A) and an erythroid-
of ALAS1 by PGC-1α is mediated by interaction of NRF-1 (nuclear specific (1B) form. In both humans and mice, the promoter region
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regulatory factor 1) and FOXO-1 (a forkhead family member) with the upstream of exon 1B contains GATA-1 sites, providing for significant
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ALAS1 promoter. When glucose levels are low, transcription of PGC- tissue-specific control of these transcripts. 47
1α is upregulated, in turn increasing ALAS1, which might precipi- The human enzyme is polymorphic with two common alleles that
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tate an attack of acute porphyria in an individual with the appropriate occur in three combinations (1–1, 1–2, and 2–2). The allele 2 sequence
inherited enzyme deficiency. Thus, upregulation of PGC-1α provides an differs from allele 1 only by a G→C transversion of nucleotide 177 in the
explanation for the induction of acute attacks of porphyria with fasting, coding region, resulting in replacement of lysine by asparagine, a more
as well as the therapeutic value of glucose loading. electronegative amino acid. ALAD exists primarily as a homooctamer.
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Regulation of heme synthesis in erythroid cells is distinct from Mutations associated with ALAD porphyria favor formation of the less
the liver. ALAS2 expression in erythroid cells is increased during ery- active hexamer. 49,50
throid differentiation when heme synthesis is increased. 33,34 Experimen- Porphobilinogen Deaminase (Hydroxymethylbilane Synthase;
tally, ALAS2 is often upregulated by heme, whereas in liver ALAS1 is Porphobilinogen Ammonia-Lyase [Polymerizing], EC 4.3.1.8) The
downregulated by heme. The β subunit of human ATP-specific succinyl fourth enzyme in the heme biosynthetic pathway catalyzes the deam-
CoA synthetase (SCS-βA) associates with human ALAS2 but not with ination and condensation of four molecules of PBG to yield the linear
ALAS1, and thereby contributes to heme synthesis in the marrow. 35 tetrapyrrole hydroxymethylbilane (HMB; see Fig. 58–4, step 3). PBG
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More than 20 ALAS2 mutations are associated with X-linked deaminase was previously known as uroporphyrinogen I synthase, and
sideroblastic anemia (Chap. 59); many are in exon 9, which contains the enzyme activity is commonly measured in the laboratory after con-
the binding site for pyridoxal 5′-phosphate (K391), and these cases are verting HMB to uroporphyrin I.
typically responsive to high doses of pyridoxine. At least one mutant This enzyme has a unique cofactor, which is a dipyrromethane
enzyme (D190V) in a patient with pyridoxine-refractory X-linked that binds the pyrrole intermediates at the catalytic site until six pyr-
sideroblastic anemia, failed to associate with SCS-βA, whereas other roles (including the dipyrrole cofactor) are assembled in a linear fash-
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ALAS2 mutants did not have this property. The mature D190V mutant ion, after which the tetrapyrrole HMB is released. The apo-deaminase
protein, but not its precursor protein, underwent abnormal processing; generates the dipyrrole cofactor to form the holo-deaminase, and this
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indicating that appropriate association of SCS-βA and ALAS2 is nec- occurs more readily from HMB than from PBG. High concentrations
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essary for functioning of ALAS2 in mitochondria. Gain-of-function of PBG may inhibit formation of the holo-deaminase.
mutations of ALAS2 have been identified in patients with XLP. 37 The gene encoding human PBG deaminase maps to chromosome
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δ-Aminolevulinate Dehydratase (Porphobilinogen Synthase; 11q23→11qter, and consists of 15 exons spread over 10 kb of DNA.
δ-Aminolevulinate Hydrolase; EC 4.2.1.24) ALA dehydratase (ALAD) Distinct erythroid-specific and housekeeping isoforms are produced
is a cytosolic enzyme that catalyzes the condensation of two molecules through alternative splicing of two distinct primary mRNA transcripts
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of ALA to form the monopyrrole PBG, with removal of two molecules arising from two promoters. The housekeeping promoter is upstream
of water (see Fig. 58–4, step 2). The enzyme functions as a homooct- of exon 1 and is active in all tissues, while the erythroid-specific pro-
amer, and requires intact sulfhydryl groups and zinc for activity. ALAD moter, which is upstream of exon 2, is active only in erythroid cells. The
activity is inhibited by sulfhydryl reagents and by lead, which displaces human housekeeping and erythroid-specific enzymes isoforms contain
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zinc. In lead poisoning (Chap. 52), erythrocyte ALAD activity is mark- 361 and 344 amino acid residues, respectively. Of the additional 17 res-
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edly inhibited, urinary ALA and coproporphyrin excretion increased, idues at the N-terminal end of the housekeeping form, 11 are encoded
erythrocyte zinc protoporphyrin elevated, and neurologic symptoms by exon 1, and 6 by a short segment of exon 3 that immediately precedes
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resemble those seen in acute porphyrias. 4,6-Dioxoheptanoic acid (suc- a methionine codon that initiates translation of the erythroid isoform.
cinylacetone) is a substrate analogue and potent inhibitor of ALAD, 41,42 Erythroid-specific trans-acting factors, such as GATA-1 and NF-E2,
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and is a byproduct of the enzyme deficiency in hereditary tyrosinemia recognize sequences in the erythroid promoter. A 1320-bp stretch of
type I. This substance is found in urine and blood of patients with this perfect identity is present between the erythroid and the nonerythroid
disease, who may also have increased ALA and symptoms resembling PBG deaminase, but with a mismatch in the first exon at their 5′ extrem-
acute porphyrias. 43 ities. An additional inframe AUG codon present 51 bp upstream from
Human ALAD mRNA has an open-reading frame of 990 bp, the initiating codon of the erythroid cDNA accounts for the additional
encoding a protein with an Mr of 36,274. Sequences known to be 17 amino acid residues at the N-terminus of the housekeeping isoform.
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essential for enzymatic activity, are those for the active site lysine res- Accordingly, a splice site mutation at the last position of exon 1, or a base
idues and for the cysteine- and histidine-rich zinc binding sites. The transition in intron 1, in certain patients with AIP results in decreased
gene for human ALAD is localized to chromosome 9p34. 45 PBG deaminase expression in nonerythroid tissues including the liver,
Studies using [ C]-ALA have shown that of the two ALA mole- but not in erythroid cells, because transcription of the gene in erythroid
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cules used as substrate, the ALA molecule contributing the propionic cells starts downstream of the site of the genetic lesion. 59
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acid side is initially bound to the enzyme. The tertiary structure of the Uroporphyrinogen III Synthase (Uroporphyrinogen III Cosyn-
yeast ALAD has been solved to 2.3-Å resolution, revealing that each thase; EC 4.2.1.75) UROS, a cytosolic enzyme, catalyzes the formation of
subunit adopts a triosephosphate isomerase barrel fold with a 39-residue uroporphyrinogen III from HMB. The process involves an intramolecular

Kaushansky_chapter 58_p0889-0914.indd 894 9/18/15 5:58 PM