ALA synthase

Author: Erika Rainero
Date: 07/02/2014


Authors: Erika Rainero, Lucia Ronco

Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. Heme serves as the prosthetic group of numerous hemoproteins (eg, hemoglobin, myoglobin, cytochromes, guanylate cyclase, and nitric oxide synthase) and plays an important role in controlling protein synthesis and cell differentiation.

Cell biology of Heme, 1999

Differences in iron metabolism and in genes for 5-aminolevulinic acid synthase (ALA-S, the first enzyme in heme biosynthesis) are responsible for the differences in regulation and rates of heme synthesis in erythroid and nonerythroid cells. In fact, there are two different genes for ALA-S, one of which is expressed ubiquitously (ALA-S1), whereas the expression of the other (ALA-S2) is specific to erythroid cells.


ALA Synthase is the committed step of the heme synthesis pathway, and is usually rate-limiting for the overall pathway.
Heme synthesis begins in mitochondria with condensation of glycine & succinyl-CoA, with decarboxylation, to form delta-aminolevulinic acid (ALA). Pyridoxal phosphate (PLP) serves as coenzyme for delta-Aminolevulinate Synthase (ALA Synthase). Condensation with succinyl-CoA takes place while the amino group of glycine is in Schiff base linkage to the aldehyde of PLP. Coenzyme A and the carboxyl of glycine are lost following the condensation reaction.
How does it work? ALA synthase removes the carboxil group from glycine and the CoA from the succinyl-CoA by means of its prosthetic group pyridoxal phosphate (a vitamin B6 derivative), forming δ-aminolevulinic acid (dALA), so called because the amino group is on the fourth carbon atom in the molecule. Glycine is initially deprotonated by a highly conserved active site lysine, leading to condensation with succinyl-CoA and loss of CoA. Protonation of the carbonyl group of the intermediate by an active site histidine leads to loss of the carboxyl group. The last intermediate is finally reprotonated to produce ALA. Dissociation of ALA from the enzyme is the rate limiting step of the enzymatic reaction and was shown to be depended upon a slow conformational change of the enzyme. The function of pyridoxal phosphate is to facilitate the removal of hydrogen, by utilizing the electrophilic pyridinium ring as an electron sink.

Amini Acids %


There are two major means of regulating the activity of the enzyme.

The first is by regulating the synthesis of the enzyme. This is important because its half-life is only about one hour.
A pilot study provides evidence for significant increasing over baseline in all subjects at 5 a.m. and 11 p.m. and of mRNA levels of ALAS1, ALAS2, PBGD at 11 p.m. in subjects with active AIP.

Circadian rythms in AIP, 2013

The second one is feedback inhibition, by an allosteric mechanism.

The lower the ATP concentration, the higher is porphyrin synthesis and ALAS activity.
The reduction of ATP is associated with an increased ALAS activity , while administration of cAMP reduces porphyrin production.

Regulation of Haemoglobin synthesis


Heme regulated degradation of delta aminolevulinate sythase in rat liver mitochondria, 2007

ALAS is an allosteric enzyme and its regulation is due to feedback inhibition by heme itself, the final product of the pathway. Heme is known to affect ALAS-1 activity by repressing gene transcription, accelerating mRNA degradation, and impeding pre-ALAS-1 mitochondrial translocation.

It is essential for ALA synthase to be incorporated into mitochondria to function physiologically, since this enzyme requires succinyl-CoA as a substrate. Kinetic studies also revealed that the transfer of ALA synthase from the liver cytosol into mitochondria is strongly inhibited by heme.
Therefore, heme acts in a novel way to prevent transport of ALAS into mitochondria, its site of function.
Furthermore, in erythroid cells, heme does inhibit cellular iron acquisition from transferrin without affecting its utilization for heme synthesis. This negative feedback is likely to explain the mechanism by which the availability of transferrin iron limits heme synthesis rate.
Moreover, in erythroid cells heme seems to enhance globin gene transcription, it is essential for globin translation, and supplies the prosthetic group for hemoglobin assembly. Heme may also be involved in the expression of other erythroid-specific proteins.

Regulation by heme of synthesis and intracellular translocation of delta-aminolevulinate synthase in the liver, 1981

Molecular regulation of 5-aminolevulinate synthase. Diseases related heme biosynthesis, 1990.

Heme also regulates ALA synthase expression post-transcriptionally by modulating mRNA stability as well as by blocking translocation of ALA synthase enzyme into the mitochondrion.

Heme regulates hepatic ALAS mRNA expressor by decreasing mRNA half-life and not by altering its role of transcription, 1991

Heme deficiency caused by several types of porphyria can causes up-regulation of hepatic 5-aminolevulinic acid synthase-1 (ALAS1) with over-production of ALA and PBG (porphobilinogen).


In developing red cells, levels of ALAS are regulated by increased gene transcription and by a post-transcriptional mechanism, in which iron most probably controls translation of erythroid ALAS mRNA through an iron-responsive element (IRE) identified in the 5' untranslated (UTR) region of the mRNA.
Because the 5'-untranslated region of the erythroid-specific ALA-S2 mRNA contains the iron-responsive element, a cis-acting sequence responsible for translational induction of erythroid ALA-S2 by iron, the availability of iron controls protoporphyrin IX levels in hemoglobin-synthesizing cells.
The inhibitory effect of activated IRPs on the synthesis of ALAS can be viewed as a way to reduce excessive protoporphirin production under condition of iron deprivation.
We can see that IRPs, binding to specific target mRNA, are able to inhibit translation of other proteins involved in iron metabolism, such as heavy (H) and light (L) subunit of ferritin and mitochondrial aconitase. On the other hand they can increase translation of transferrin receptor (TfR1) to improve iron uptake.

Molecular control of iron metabolism, 1996


Hepcidin is a peptide hormone produced by the liver. Hepcidin functions to regulate (inhibit) iron transport across the gut mucosa, thereby preventing excess iron absorption and maintaining normal iron levels within the body. Hepcidin inhibits iron transport by binding to the iron export channel ferroportin, which is located on the basolateral surface of gut enterocytes and the plasma membrane of reticuloendothelial cells (macrophages). Inhibiting ferroportin prevents iron from being exported and the iron is sequestered in the cells. By inhibiting ferroportin, hepcidin prevents enterocytes of the intestines from secreting iron into the hepatic portal system, thereby functionally reducing iron absorption. Therefore, the hepcidin maintains iron homeostasis.



Steroid hormones cause changes within a cell by first passing through the cell membrane of the target cell. Steroid hormones, unlike non-steroid hormones, can do this because they are fat-soluble. Cell membranes are composed of a phospholipid bilayer which prevents fat-insoluble molecules from diffusing into the cell.

Once inside the cell the steroid hormone binds with a specific receptor found only in the cytoplasm. The receptor bound steroid hormone then travels into the nucleus and binds to another specific receptor on the chromatin, where there are Hormone Responsive Elements (HRE). Once bound to the chromatin, this steroid hormone-receptor complex induces the production of messenger RNA (mRNA) molecules (transcription). The mRNA molecules are then modified and transported to the cytoplasm. The mRNA molecules code for the production of proteins through a process called translation.

Lots of experiments demonstrated that the administration of estrogens (mainly estradiol) increased hepatic sintesis of ALA synthase. Really they cause an initial decline of hepatic ALA synthase and this would appear somewhat paradoxical, since evidence previously presented suggests that estrogen may be an inducer of hepatic ALA synthase. This previous studies utilized techniques which do not provide instantaneous values of enzyme level, but rather are measures of the integral of enzyme activity over a period of time. The initial decline of hepatic ALA synthase following estrogens indicates that it may not be a primer inducer of the enzyme, so induction appears to occur by a rebound mechanism.

Also estrogens administered as oral contraceptives (OCs) increase the activity of ALA synthase, then they can provoke acute intermittent porphyria (AIP).

Oscillations of hepatic ALAS produced by estrogens, 1967

Certain natural steroid metabolites significantly stimulate erythropoiesis in normal human bone marrow cells in culture.

The influence of steroid hormone metabolites on the in vitro development of erythroid colonies derived from human bone marrow, 1979

This induction is because different cytochromes P450 are involved in steroid biosynthesis. These cytochromes have heme as the prosthetic group.


Hemin is an iron-containing porphyrin. More specifically, it is Protoporphyrin IX containing a ferric iron ion (Heme B) with a chloride ligand.
Low concentrations of exogenous hemin (30-35 microM) inhibited the biosynthetic labelling of mature erythroid ALA synthase. Parallel experiments using antibodies directed against human H-chain ferritin confirmed the specificity of the effects of hemin on translation of the e-ALA synthase mRNA and its degradation. Although the mechanism of hemin action is unknown, it is apparently independent of 5'-iron-response elements and influences the translational activity of erythroid ALA synthase mRNA.


5-Aminolevulinate synthase is a pyridoxal 5'-phosphate-dependent enzyme and is functional as a homodimer. Pyridoxal 5'-phosphate is bound to Lysine 313. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA-derived 5-aminolevulinate synthase sequences and is present in the C-terminal (catalytic) domain. Mutagenesis of the 5-aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5'-phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein. Pyridoxine deficiency can lead to a sideroblastic anemia.


Phenobarbital treatment of rat cultures increased the total amount of cytochrome P450 and ALA-S activity and ALA-S mRNA. Treatment with phenobarbital combined with succinyl acetone synergistically increased both ALA-S activity and ALA-S mRNA, presumably by blocking formation of heme, the feedback repressor of ALA-S. Indeed, the increase in ALA-S mRNA caused by the combined treatment was abolished by adding heme itself to the cultures.
Barbiturates, by causing ALA synthase activity increase, induce high levels of ALA and PBG in urine and an accumulation of protoporphyrin in liver, leading to regressive processes and cirrhosis.

Role of Heme in phenobarbital induction of ALAS in cultured rat hepatocytes, 1990.


Hepatocyte heme production must be controlled to respond to changing metabolic requirements. As we said, hepatocytes express ALAS-1 and increasing heme levels create a negative feedback that downregulate transcription of ALAS-1 and inhibit its import into the mitochondrial matrix. ALAS-1 transcription is upregulated by peroxisome proliferator-activated receptor coactivator 1 (PGC-1). Transcription of PGC-1 is regulated by glucose levels. Hypoglycemia induces PGC-1 production, increasing ALAS-1 and heme synthesis. This promotes the clinical appearance of the acute porphyrias. Moreover, PGC-1 increases mitochondrial genes expressions, such as oxidative respiration genes, it increases fatty acid oxidation and glucose uptake by GLUT 4.

Biosynthesis of heme in mammals, 2006

Glucagon and dibutyryl CAMP enhanced the induction of ALA synthase suggesting that the glucose effect may be mediated by changes in CAMP levels. These data may be relevant for the treatment with glucose and heme of patients with “inducible” hepatic porphyria.

Glucagon, cAMP and ALA synthase,1981

The red blood cell form is coded by a gene on chromosome X, whereas the other form is coded by a gene on chromosome 3. The disease X-linked sideroblastic anemia is caused by mutations in the ALA synthase gene on chromosome X, whereas no diseases are known to be caused by mutations in the other gene. Gain of function mutations in the erythroid specific ALA synthase gene have been shown recently to cause a previously unknown form of porphyria known as X-linked-dominant protoporphyria.

Sideroblastic anemia

ALA (aminolevulinate) is toxic to the brain. This may be due in part to the fact that ALA is somewhat similar in structure to the neurotransmitter GABA. In addition, autoxidation of ALA generates reactive oxygen species (oxygen radicals).

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