Synthesis of Heme

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Synthesis of Heme
Contents of this page:
Heme
Synthesis of d-aminolevulinate & porphobilinogen
Formation & modification of the tetrapyrrole ring system
Porphyrias

Heme is the prosthetic group of hemoglobin, myoglobin, and the cytochromes. The heme of cytochrome c is shown at right. (For the slightly different structure of heme a, see the notes on electron transfer.) Heme is an asymmetric molecule. (Note the positions of the methyl side chains around the ring system.)
The heme ring system is synthesized from glycine and succinyl-CoA.

Using isotopic tracers, it was initially found that N & C atoms of heme are derived from glycine and acetate. It was later determined that the labeled acetate first enters Krebs Cycle as acetyl-CoA, and the labeled carbon becomes incorporated into succinyl-CoA, which is the more immediate precursor of heme.

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/images/hemecytc.gif



Heme synthesis begins with condensation of glycine & succinyl-CoA, with decarboxylation, to form d-aminolevulinic acid (ALA).

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/ala.gif


Pyridoxal phosphate (PLP) serves as coenzyme for d-Aminolevulinate Synthase. The enzyme is evolutionarily related to transaminases.
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/plpsm.gif

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. Diagram p. 1015.
d-Aminolevulinate Synthase (ALA Synthase) is the committed step of the heme synthesis pathway, and is usually rate-limiting for the overall pathway. The amount of the enzyme is regulated through control of gene transcription. Heme functions as a feedback inhibitor, repressing transcription of the gene for d-Aminolevulinate Synthase in most cells.
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/glplp.gif

PBG Synthase (Porphobilinogen Synthase), also called ALA Dehydratase, catalyzes condensation of two molecules of d-aminolevulinic acid (ALA) to form porphobilinogen (PBG).

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/pbgsynth.gif

The reaction mechanism involves two lysine residues and a bound cation at the active site. The bound cation in the mammalian enzyme is Zn++.
As each of the two d-aminolevulinate (ALA) substrates binds at the active site, its keto group initially reacts with the side-chain amino group of one of the two lysine residues to form a Schiff base. These Schiff base linkages promote the C-C and C-N condensation reactions that follow, assisted by the metal ion that coordinates to the ALA amino groups.

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/alaschiff.gif

The binding sites for Zn++ in the homo-octomeric mammalian Porphobilinogen Synthase, which include cysteine S ligands, can also bind Pb++ (lead). Inhibition of Porphobilinogen Synthase by Pb++ results in elevated blood ALA, which may cause some of the neurological effects of lead poisoning.
ALA (d-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 (g-aminobutyric acid). In addition, reactive oxygen species (oxygen radicals) are generated during autoxidation of ALA.
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/gaba.gif

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Current views of the reaction mechanism are based on solved crystal structures of:
a bacterial PBG Synthase with a substrate analog in Schiff base linkage at each of two binding sites for ALA.
a yeast PBG Synthase crystallized in presence of the ALA substrate and having at its active site an intermediate resembling the product PBG still in Schiff base linkage to one lysine side-chain.
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/images/chimeatp.gif http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/images/chimeatp.gif

Explore these structures at right.

PBG Synthase - ALA
PBG Synthase - PBG

Porphobilinogen is the first pathway intermediate that includes a pyrrole ring.

The porphyrin ring is formed by condensation of four molecules of porphobilinogen (PBG).

Porphobilinogen Deaminase catalyzes successive condensations of PBG, initiated in each case by elimination of the amino group. Diagram p. 1018.

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/pyrrole.gif http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/pbg.gif

Porphobilinogen Deaminase enzyme has a dipyrromethane prosthetic group, linked at the active site via a cysteine S.
The enzyme catalyzes formation of its own dipyrromethane prosthetic group by condensation of 2 molecules of PBG.
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/dipyrromet.gif


Once four PBGs have condensed, prior to hydrolysis of the link to the enzyme's dipyrromethane, the enzyme has a bound hexapyrrole, derived from 6 PBG.

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/hmbprosth.gif


Porphobilinogen Deaminase is organized in 3 domains. Predicted interdomain flexibility may accommodate the growing polypyrrole in the active site cleft.


Hydrolysis of the link to the dipyrromethane yields free hydroxymethylbilane

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/hmb.gif


.

Explore at right the structure of the enzyme Porphobilinogen Deaminase, with its covalently linked prosthetic group dipyrromethane.
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/images/chimeatp.gif


Uroporphyrinogen III Synthase converts the linear tetrapyrrole hydroxymethylbilane to the macrocyclic uroporphyrinogen III.
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/prepost.gif

Uroporphyrinogen III Synthase catalyzes ring closure, and flipping over one of the pyrroles, to yield an asymmetric tetrapyrrole. Note the distribution of acetyl and propionyl side chains in the diagram above.
This rearrangement is thought to proceed via a spiro intermediate, as depicted at right and in the animation below.

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/spiro.gif


The active site of Uroporphyrinogen III Synthase is located in a cleft between two domains of the enzyme. The structural flexibility inherent in this arrangement is proposed to be essential to catalysis.
Uroporphyrinogen III is the precursor for synthesis of vitamin B12, chlorophyll, and heme, in organisms that produce these compounds.
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/upgsynth.gif



Conversion of uroporphyrinogen III to protoporphyrin IX (above) occurs in several steps, as presented in the animation below.
These steps include:


decarboxylation of all 4 acetyl side chains, converting them to methyl groups
oxidative decarboxylation of 2 of the 4 propionyl side chains, converting them to vinyl groups
oxidation to add more double bonds, catalyzed by Protoporphyrinogen Oxidase.
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/upgppix.gif


Fe++ is added to protoporphyrin IX via Ferrocheletase, to yield heme.
Fe++ may bind to a conserved histidine residue in the enzyme, prior to being transferred to protoporphyrin IX.
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/protoporph.gif

Porphyrias are genetic diseases, in each of which activity of one of the enzymes involved in heme synthesis is decreased. Symptoms vary depending on the enzyme, the severity of the deficiency and whether heme synthesis is affected primarily in liver or in developing erythrocytes.

Acute hepatic porphyrias are associated with occasional episodes of severe neurological symptoms. Permanent nerve damage and even death can result, if not treated promptly. Elevated d-aminolevulinic acid (ALA), arising from de-repression of ALA Synthase gene transcription when heme is deficient, is considered responsible for the neurological symptoms. Diagnosis is based in part on elevated levels of porphyrin precursors, e.g., porphobilinogen and/or d-aminolevulinic acid, in urine.

Treatment of episodes of acute hepatic porphyria is by injection of hemin (a form of heme). The heme, in addition to supplying needs, would repress transcription of the gene for ALA Synthase, which is rate-limiting for the pathway and the source of the excess ALA.

For more information on these diseases, search the OMIM (Online Mendelian Inheritance in Man) website with the keyword porphyria.

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/animat.gif