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Synthesis of heme
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Dr.Ehab Aboueladab

Dr.Ehab Aboueladab

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  • 1. Synthesis of Heme
  • 2. CH3 CH3 HC S CH2 protein N H3C CH3 N Fe N − protein OOC CH2 CH2 CH S CH2 N CH3 CH2 CH3 CH2 COO− Heme cHeme is the prosthetic group of hemoglobin, myoglobin, &cytochromes. Heme is an asymmetric molecule. E.g., note thepositions of methyl side chains around the ring system.
  • 3. The heme ring system is synthesized from glycine &succinyl-CoA.Using isotopic tracers, it was initially found that N & Catoms of heme are derived from glycine and acetate.It was later determined that the labeled acetate entersKrebs Cycle as acetyl-CoA, and the labeled carbonbecomes incorporated into succinyl-CoA, the moreimmediate precursor of heme.
  • 4. O− − OOC CH2 CH2 C S-CoA + OOC CH2 NH3+ succinyl-CoA H+ glycineδ-AminolevulinicAcid Synthase CoA-SH CO2 O − OOC CH2 CH2 C CH2 NH3+ δ-aminolevulinate (ALA)Heme synthesis begins with condensation ofglycine & succinyl-CoA, with decarboxylation,to form δ-aminolevulinic acid (ALA).
  • 5. H OPyridoxal phosphate −O O− C H2(PLP) serves as coenzyme P C OHfor δ-Aminolevulinate O OSynthase (ALA Synthase), +an enzyme evolutionarily N CH3 Hrelated to transaminases. Pyridoxal phosphate (PLP)Condensation withsuccinyl-CoA takes place H2C COO−while the amino group of N+glycine is in Schiff base O− HC H −O H2linkage to the PLP aldehyde. P C O− OCoA & the glycine Ocarboxyl are lost following + N CH3the condensation. H glycine-PLP Schiff base (aldimine)
  • 6. ALA Synthase is the committed step of the hemesynthesis pathway, & is usually rate-limiting for theoverall pathway.Regulation occurs through control of gene transcription.Heme functions as a feedback inhibitor, repressingtranscription of the ALA Synthase gene in most cells.A variant of ALA Synthase expressed only in developingerythrocytes is regulated instead by availability of iron inthe form of iron-sulfur clusters.
  • 7. PBG Synthase COO− COO− COO− CH2 CH2 COO− CH2 CH2 CH2 2 H 2O CH2 CH2 + C O C O C C CH2 CH2 H2C C CH N + + + NH3 NH3 NH3 H 2 δ-aminolevulinate porphobilinogen (ALA) (PBG)PBG Synthase (Porphobilinogen Synthase), alsocalled ALA Dehydratase, catalyzes condensation oftwo molecules of δ-aminolevulinate to form thepyrrole ring of porphobilinogen (PBG).
  • 8. The reaction HOOCmechanism involves CH2 CH2 Lys−Enzyme2 Lys residues & a H2C CH2 CH2bound cation at the +active site. C NH H2CCation in mammalian ALA in Schiff base linkage NH2 to active site lysineenzyme is Zn++.As each of the two δ-aminolevulinate (ALA) substratesbinds at the active site, its keto group initially reacts withthe side-chain amino group of one of the two lysineresidues to form a Schiff base.These Schiff base linkages promote the C-C and C-Ncondensation reactions that follow, assisted by the metalion that coordinates to the ALA amino groups.
  • 9. HOOC COO− CH2 CH2 Lys−Enzyme COO− CH2H2C CH2 CH2 CH2 CH2 + C NHH2C ALA in Schiff base linkage H2C NH2 to active site lysine N + H NH3 A proposed reaction mechanism Porphobilinogen (PBG) is based on crystal structures of:  a bacterial PBG Synthase with a substrate analog in Schiff base linkage at each of 2 ALA binding sites.  a yeast PBG Synthase crystallized with ALA substrate, having at its active site an intermediate resembling PBG in Schiff base linkage to one lysine side-chain.
  • 10. The Zn++ binding sites in the homo-octomeric mammalianPorphobilinogen Synthase, which include cysteine Sligands, can also bind Pb++ (lead).Inhibition of Porphobilinogen Synthase by Pb++ results inelevated blood ALA, as impaired heme synthesis leads tode-repression of transcription of the ALA Synthase gene.High ALA is thought to cause some of the neurologicaleffects of lead poisoning, although Pb++ COO− COO−also may directly affect the nervous system. CH2 CH2ALA is toxic to the brain, perhaps due to: CH2 CH2• Similar ALA & neurotransmitter GABA C O CH2 (γ-aminobutyric acid) structures. CH2 NH3+• ALA autoxidation generates reactive NH3+ oxygen species (oxygen radicals). ALA GABA
  • 11. COO− COO− CH2 CH2 CH2 Porphobilinogen (PBG) is the first H2C N pathway intermediate N + H H that includes a NH3 pyrrole pyrrole ring. Porphobilinogen (PBG)The porphyrin ring is formed by condensation of 4molecules of porphobilinogen.Porphobilinogen Deaminase catalyzes successive PBGcondensations, initiated in each case by elimination ofthe amino group.
  • 12. COO - COO- COO - CH2 COO- CH2 CH2 CH2 CH2 CH2Enz S N N H H dipyrromethanePorphobilinogen Deaminase has a dipyrromethaneprosthetic group, linked at the active site via a cysteine S.The enzyme itself catalyzes formation of this prostheticgroup.
  • 13. COO- CH2 COO- COO- COO- COO-CH CH2 2 COO- CH2 COO- CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 COO- NH HNEnz S N N NH HN H H CH2 COO- CH2 CH2 CH2 CH2 COO-COO- CH2 COO-PBG units are added to the dipyrromethane until a linearhexapyrrole has been formed.
  • 14. COO- CH2 COO-Porphobilinogen CH2 CH2Deaminase isorganized in 3 - OOC CH2 CH2 CH2 COO-domains. NH HNPredicted HO NH HNinterdomain CH2 COO-flexibility may CH2accommodatethe growing CH2 CH2 CH2polypyrrole in the COO-COO- CH2active site cleft. hydroxymethylbilane COO-Hydrolysis of the link to the enzymes dipyrromethanereleases the tetrapyrrole hydroxymethylbilane.
  • 15. hydroxy- COO- COO- uroporphyrinogenmethylbilane III CH2 COO- CH2 COO - CH2 CH2 CH2 CH2-OOC CH2 CH2 CH2 COO- - OOC CH2 CH2 CH2 COO- NH HN NH HN HO C NH HN C NH HN CH2 COO- - OOC CH2 CH2 COO- C C CH2 CH2 CH2 CH2 CH2 CH2 COO-COO- CH2 CH2 CH2 Uroporphyrinogen III COO- Synthase COO- COO- Uroporphyrinogen III Synthase converts the linear tetrapyrrole hydroxymethylbilane to the macrocyclic uroporphyrinogen III.
  • 16. Uroporphyrinogen COO-III Synthase CH2 COO-catalyzes ring CH2 CH2closure & flippingover of one pyrrole - OOC CH2 CH2 CH2 COO-to yield an HN NHasymmetrictetrapyrrole. N C HN HC CH2 COO-This rearrangementis thought to CH2proceed via a spiro CH2 COO- CH2intermediate. CH2 CH2 postulated COO- COO- intermediate
  • 17. hydroxy- COO- COO- uroporphyrinogenmethylbilane III CH2 COO- CH2 COO - CH2 CH2 CH2 CH2-OOC CH2 CH2 CH2 COO- - OOC CH2 CH2 CH2 COO- NH HN NH HN HO C NH HN C NH HN CH2 COO- - OOC CH2 CH2 COO- C C CH2 CH2 CH2 CH2 CH2 CH2 COO-COO- CH2 CH2 CH2 Uroporphyrinogen III COO- Synthase COO- COO- Note the distribution of acetyl & propionyl side chains, as flipping over of one pyrrole yields an asymmetric tetrapyrrole.
  • 18. The active site ofUroporphyrinogen IIISynthase is in a cleft betweentwo domains of the enzyme.The structural flexibilityinherent in this arrangementis proposed to be essential tocatalysis.Uroporphyrinogen III is the precursor for synthesis ofvitamin B12, chlorophyll, and heme, in organisms thatproduce these compounds.Conversion of uroporphyrinogen III to protoporphyrin IXoccurs in several steps.
  • 19. - COO uroporphyrinogen III protoporphyrin IX CH2 COO- CH2 CH2 CH2 CH CH3- - H3C CH CH2 OOC CH2 CH2 CH2 COO NH HN NH N NH HN N HN- -OOC CH2 CH2 COO H3C CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 COO- COO- COO - COO -  All 4 acetyl side chains are decarboxylated to methyl groups (catalyzed by Uroporphyrinogen Decarboxylase)  Oxidative decarboxylation converts 2 of 4 propionyl side chains to vinyl groups (catalyzed by Coproporphyrinogen Oxidase)  Oxidation adds double bonds (Protoporphyrinogen Oxidase).
  • 20. CH2 protoporphyrin IX CH2 heme CH CH3 CH CH3H3C CH CH2 H3C CH CH2 NH N N N Fe++ 2H+ Fe N HN N NH3C CH3 H3C CH3 CH2 CH2 Ferrochelatase CH2 CH2 CH2 CH2 CH2 CH2 COO- COO - COO- COO-Fe++ is added to protoporphyrin IX via Ferrocheletase, ahomodimeric enzyme containing 2 iron-sulfur clusters.A conserved active site His, along with a chain of anionicresidues, may conduct released protons away, as Fe++ bindsfrom the other side of the porphyrin ring, to yield heme.
  • 21. Regulation of transcription or post-translational processingof enzymes of the heme synthesis pathways differs betweenerythrocyte forming cells & other tissues.  In erythrocyte-forming cells there is steady production of pathway enzymes, limited only by iron availability.  In other tissues expression of pathway enzymes is more variable & subject to feedback inhibition by heme.Porphyrias are genetic diseases in which activity of oneof the enzymes involved in heme synthesis is decreased(e.g., PBG Synthase, Porphobilinogen Deaminase, etc…).Symptoms vary depending on  the enzyme  the severity of the deficiency  whether heme synthesis is affected primarily in liver or in developing erythrocytes.
  • 22. Occasional episodes of severe neurological symptoms areassociated with some porphyrias. Permanent nerve damage and even death can result, if not treated promptly. Elevated δ-aminolevulinic acid (ALA), arising from de-repression of ALA Synthase gene transcription, is considered responsible for the neurological symptoms.Photosensitivity is another common symptom. Skin damage may result from exposure to light. This is attributable to elevated levels of light-absorbing pathway intermediates and their degradation products.
  • 23. Question: How do you think episodes of acute neurologicalsymptoms would be treated?Treatment is by injection of hemin (a form of heme).Why would this work?The heme, in addition to supplying needs, would represstranscription of the gene for ALA Synthase, rate-limitingfor the pathway and the source of excess ALA.View an amination of the heme synthesis pathway.
  • 24. Regulation of iron absorption & transport.Iron for synthesis of heme, Fe-S centers & othernon-heme iron proteins is obtained from: the diet release of recycled iron from macrophages of the reticuloendothelial system that ingest old & damaged erythrocytes.There is no mechanism for iron excretion. Iron is significantly lost only by bleeding, including menstruation in females. Small losses occur from sloughing of cells of skin & other epithelia.
  • 25. transferrin with bound Fe extracellular space transferrin receptor receptor-mediated endocytosisIron is transported in blood serum bound to the proteintransferrin.The plasma membrane transferrin receptor mediatesuptake of the complex of iron with transferrin by cellsvia receptor mediated endocytosis.
  • 26. Iron is stored within cells as a complex with the proteinferritin.The main storage site is liver.The plasma membrane protein ferroportin mediates: release of absorbed iron from intestinal cells to blood serum release of iron from hepatocytes (liver cells) and macrophages.Control of dietary iron absorption and serum ironlevels involves regulation of ferroportin expression.
  • 27.  Transcription of the gene for the iron transporter ferroportin is responsive to iron. Hepcidin, a regulatory peptide secreted by liver, induces degradation of ferroportin. Hepcidin secretion increases when iron levels are high or in response to cytokines produced at sites of inflammation. Degradation of ferroportin leads to decreased absorption of dietary iron and decreased serum iron. Hepcidin is considered an antimicrobial peptide because by lowering serum iron it would limit bacterial growth.
  • 28. Hereditary hemochromatosis is a family of geneticdiseases characterized by excessive iron absorption,transport & storage.Genes mutated in these disorders include those for: transferrin receptor a protein HFE that interacts with transferrin receptor hepcidin hemojuvelin, an iron-sensing protein required for transcription of the gene for hepcidin.E.g., impaired synthesis or activity of hepcidin leads tounrestrained ferroportin activity, with high dietary intakeand high % saturation of serum transferrin with iron.Organs particularly affected by accumulation of excessiron include liver and heart.