What are the functions of heme?
-
Transport of oxygen (hemoglobin, myoglobin)
-
Electron transport (respiratory cytochromes)
-
Oxidation-reduction reactions (cytochrome P450 enzymes)
- Where are the major sites of heme synthesis?
- Where else is heme synthesized?
- What cannot synthesize heme?
- Major Sites:
-
bone marrow ⇒ hemoglobin (6-7g hemoglobin are synthesized each day)
-
liver ⇒ cytochrome P450 enzymes (drug detoxificaton)
- However, heme is also required for other important cellular proteins and is synthesized in virtually all cells,
-
mature erythrocytes do not synthesize heme (lack mitochondria)
- bone marrow ⇒ hemoglobin (6-7g hemoglobin are synthesized each day)
- liver ⇒ cytochrome P450 enzymes (drug detoxificaton)
- What are porphyrins?
- What is the structure of heme?
-
Porphyrins: cyclic tetrapyrroles capable of chelating to various metals to form essential prosthetic groups for various biological molecules
- Heme is predominantly a planar molecule
- porphyrin derrivative + a single ferrous ion (Fe2+ = reduced form of iron)
- porphyrin derrivative + a single ferrous ion (Fe2+ = reduced form of iron)
Heme = ?
- What is heme oxidized to?
Heme = Ferroprotoporphyrin IX
- Ferroprotoporphyrin IX (heme) is rapidly autooxidized to ferriprotoporphyrin IX ("hemin"; contains ferric Fe3+ iron)
7 major steps of heme biosynthesis:
- The 1st and last 3 steps occur in the ....
- The intermediate steps occur in the ....
- The 1st and last 3 steps occur in the mitochondrion
- The intermediate steps occur in the cytosol
What is the committed step of heme synthesis?
Step 1: condensation of glycine and succinyl-CoA with decarboxylation, to yield 5-aminolevulinate (ALA)
Step 1 of heme synthesis:
- Reaction:
- Enzyme:
- Location:
- Cofactor:
-
Reaction: condensation of glycine and succinyl-CoA with decarboxylation, to yield 5-aminolevulinate (ALA)
-
Enzyme: 5-aminolevulinate synthase (ALAS)
-
Location: ALAS is localized to the inner mitochondrial membrane
- encoded by a nuclear gene family
- must be imported into the mitochondrion
-
Cofactor: pyridoxal phosphate (PLP) dependent enzyme (vitamin B6)
- Condensation with succinyl-CoA takes place while the amino group of glycine is in Schiff base linkage to the PLP aldehyde
- encoded by a nuclear gene family
- must be imported into the mitochondrion
- Condensation with succinyl-CoA takes place while the amino group of glycine is in Schiff base linkage to the PLP aldehyde
What are the isoforms of ALAS?
Two isoforms of ALAS:
- ALAS1 is the liver isoform
- ALAS2 is the erythroid/reticulocyte isoform
Describe the regulation of ALAS1:
-
Feedback inhibition by heme or hemin regulates heme biosynthesis in the liver
-
Heme (hemin) exerts multiple regulatory effects on hepatic heme biosynthesis by inhibiting ALAS1 synthesis at both transcriptional and translational levels, as well as its mitochondrial import
- drugs or metabolites can increase ALAS1 activity
-
increase the synthesis of cytochrome P450 enzymes ⇒ increasing the demand for heme
- increase the synthesis of cytochrome P450 enzymes ⇒ increasing the demand for heme
Describe the regulation of ALAS2:
- Heme biosynthesis in erythroid cells is NOT regulated by feedback repression of ALAS2 by heme
- In reticulocytes (immature RBCs), heme stimulates synthesis of globin and ensures that heme & globin are synthesized in the correct ratio for assembly into hemoglobin
- Drugs that cause a marked elevation in ALAS1 activity, such as phenobarbital, do not affect ALAS2
Step 2 of heme biosynthesis:
- Reaction:
- Enzyme:
- Cofactor:
- Location:
- Complication:
-
Reaction: condensation of two molecules of ALA to form one molecule of porphobilinogen (PBG)
-
first pathway intermediate that includes a pyrrole ring
-
Enzyme: ALA dehydratase (ALAD)
-
Cofactor: Zn2+
- lead and other heavy metals can displace the Zn2+ and eliminate catalytic activity
-
Location: cytosol
-
Complication: lead poisoning
- increase ALA in urine
- clinical manifestations that mimic acute porphyrias
- first pathway intermediate that includes a pyrrole ring
- lead and other heavy metals can displace the Zn2+ and eliminate catalytic activity
- increase ALA in urine
- clinical manifestations that mimic acute porphyrias
Effects of lead poisoning:
- Heme synthesis:
- Neurologic symptoms:
-
Inhibition of ALA dehydratase (aka, porphobilinogen synthase) by lead (Pb2+) results in elevated blood ALA
- as impaired heme synthesis leads to de-repression of transcription of the ALAS gene
-
ALA is toxic to the brain, perhaps due to:
-
Similar ALA & neurotransmitter GABA (γ-aminobutyric acid) structures
-
ALA autoxidation generates reactive oxygen species (ROS)
- as impaired heme synthesis leads to de-repression of transcription of the ALAS gene
- Similar ALA & neurotransmitter GABA (γ-aminobutyric acid) structures
- ALA autoxidation generates reactive oxygen species (ROS)
Step 3 of heme synthesis:
- Reaction:
- Enzyme:
- Coenzyme:
- Location:
-
Reaction:
-
Step 1: head-to-tail condensation of 4 porphobilinogen molecules to form hydroxymethylbilane (linear tetrapyrrole)
- Each condensation ⇒ liberation of one ammonium ion
-
Step 2: hydroxymethylbilane ⇒ uroporphyrinogen III
-
Enzyme: porphobilinogen deaminase (PBGD) or uroporphyrinogen I synthase
-
Coenzyme: uroporphyrinogen III cosynthase
-
Location: cytosol
-
Step 1: head-to-tail condensation of 4 porphobilinogen molecules to form hydroxymethylbilane (linear tetrapyrrole)
- Each condensation ⇒ liberation of one ammonium ion
- Step 2: hydroxymethylbilane ⇒ uroporphyrinogen III
What is the role of uroporphrinogen III cosynthase?
- The tetrapyrrole can spontaneously cyclize to form uroporphyrinogen I (nonenzymatic) which IS NOT in the normal pathway for heme biosynthesis
- However, PBGD is tightly associated with a second enzyme uroporphyrinogen III cosynthase (UROS)
- no enzymatic activity alone
- serves to direct the stereochemistry of the condensation reaction to yield the uroporphyrin ogen III isomer which IS on the pathway for heme biosynthesis
- no enzymatic activity alone
- serves to direct the stereochemistry of the condensation reaction to yield the uroporphyrin ogen III isomer which IS on the pathway for heme biosynthesis
Step 4 of heme synthesis:
- Reaction:
- Enzyme:
- Location:
-
Reaction: uroporphyrinogen III ⇒ coprophorphyrinogen III
- decarboxylation of acetate side chains to methyl groups
-
Enzyme: Uroporphyrinogen decarboxylase (UROD)
-
Location: cytosol
- decarboxylation of acetate side chains to methyl groups
Step 5 of heme synthesis:
- Reaction:
-
Enzyme:
- What is being converted?
-
Location:
- What does this imply?
-
Reaction: Coproporphyrinogen III ⇒ protoporphyrinogen IX
- transported into the intermembrane space
-
Enzyme: coproporphyrinogen III oxidase (CPO)
- converts specific propionic acid side chains to vinyl groups
-
Location: intermembrane space of the mitochondrion
- implying that its product or protoporphyrin IX must cross the inner mitochondrial membrane because heme is formed within the inner membrane
- transported into the intermembrane space
- converts specific propionic acid side chains to vinyl groups
- implying that its product or protoporphyrin IX must cross the inner mitochondrial membrane because heme is formed within the inner membrane
Step 6 of heme synthesis:
- Reaction:
- Enzyme:
- Location:
-
Reaction: protoporphyrinogen IX ⇒ protoporphyrin IX (moving double bonds)
-
Enzyme: protoporphyrinogen IX oxidase (PPO)
-
Location: mitochondrion
Step 7 of heme synthesis:
- Reaction:
- Enzyme:
- Location:
-
Reaction: Insertion of Fe2+ into protoporphyrin IX to generate HEME
-
Enzyme: ferrochelatase
-
Location: mitochondrion
- What can inhibit Step 7 of heme synthesis?
- What will result in a brillant flourescent complex?
-
Ferrochelatase is inhibited by lead (lead poisoning; increase protoporphyrin in urine) and is also inhibited during iron deficiency (anemia)
-
In the absence of Fe2+:
-
ferrochetalase can insert Zn2+ into the protoporphyrin ring to yield a brilliantly fluorescent complex
- ferrochetalase can insert Zn2+ into the protoporphyrin ring to yield a brilliantly fluorescent complex
What are porphyrias?
inherited genetic or acquired (rarely) disorders resulting from deficiency in specific enzymes of the porphyrin/heme biosynthetic pathway
- How are porphyrias classified?
- What is the pattern of inheritance?
Either hepatic or erythroid
- reflect the principal sites of heme biosynthesis
- depend on the site of expression of the enzyme defect
- Inheritance: autosomal dominant
- Except congenital erythropoietic porphyria (autosomal recessive)
- What causes symptoms seen in porphyrias?
- What is the difference in location of the defect (early vs. late)?
-
Accumulation of intermediates upstream from the enzyme defect results in the clinical symptoms associated with the various porphyrias
-
Defects early in the biosynthetic pathway (accumulation of ALA, prophobilinogen) result in neurologic dysfunction
-
Defects later in the pathway (accumulation of cyclic tetrapyrroles, but not prophobilinogen) result in sunlight-induced cutaneous lesions:
- in the presence of molecular oxygen, UV irradiation of cyclic tetrapyrroles generates reactive oxygen species that can produce cellular damage
- in the presence of molecular oxygen, UV irradiation of cyclic tetrapyrroles generates reactive oxygen species that can produce cellular damage
What are the acute porphyrias?
- Definiton:
- Symptoms:
- Examples:
- Periodic acute attacks
- Symptoms: abdominal pain, neurologic deficits, psychiatric symptoms, and reddish-colored urine.
- Examples:
- Doss porphyria (ALA dehydratase deficiency)
- Acute intermittent porphyria
- Hereditary coproporyphyria
- Variegate porphyria
- Doss porphyria (ALA dehydratase deficiency)
- Acute intermittent porphyria
- Hereditary coproporyphyria
- Variegate porphyria
What are chronic porphyrias?
-
Dermatologic diseases that may or may not include the liver and nervous system
- Examples:
- Congenital erythropoietic porphyria (Gunther's disease)
- Erythropoietic porphyria/protoporphyria
- Porphyria cutanea tarda
- Congenital erythropoietic porphyria (Gunther's disease)
- Erythropoietic porphyria/protoporphyria
- Porphyria cutanea tarda
- What enzymes are particularly sensitive to lead poisoning?
- What will be seen in the urine during lead poisoning?
-
Ferrochelatase and ALA dehydratase are particularly sensitive to lead poisoning
-
Protoporphyrin and ALA accumulate in the urine
What is the function of hemoglobin?
- Hemoglobin is a specialized protein designed to transport oxygen (O2) from the lungs, a region of high O2 concentration, to peripheral tissues, where oxygen is low
- Metabolism in the peripheral tissues generates CO2 and H+ that are transported back to the lungs, in part, by hemoglobin.
-
O2 has very low solubility in plasma
- As a consequence, >98% of the O2 that reaches tissues is carried in red blood cells (RBCs) bound to Hemoglobin
How is CO2 transport different from O2 transport?
-
RBCs contain carbonic anhydrase which catalyzes the rapid reversible hydration of CO2 to carbonic acid (H2CO3).
- H2CO3 then rapidly and spontaneously dissociates to bicarbonate (HCO3-) and a H+
- CO2 and HCO3- are soluble in plasma and RBC cytosol
- most of the CO2 made in tissues returns to the lungs as those species
- about 14% of the CO2 made is carried bound to Hb
- most of the CO2 made in tissues returns to the lungs as those species
- about 14% of the CO2 made is carried bound to Hb
- What is the structure of hemoglobin?
- What is hemoglobin related to?
- Which form of Fe can bind O2?
- Which form of iron cannot bind O2? What is it called?
-
Hemoglobin is a heterotetrameric protein (αβ)2
- Both subunits are evolutionarily related to myoglobin
- a monomeric protein abundant in muscle that is designed to store O2
- myoglobin: 1 heme groups
- hemoglobin: 4 heme groups
-
Fe2+ is the ferrous form of iron that is capable of binding O2
-
Fe3+ is the ferric form of iron that CANNOT bind O2 and is present in an INACTIVE form
-
methemoglobin (metHb)
- a monomeric protein abundant in muscle that is designed to store O2
- myoglobin: 1 heme groups
- hemoglobin: 4 heme groups
- methemoglobin (metHb)
Describe the cooperative binding curve for oxygen:
-
Myoglobin gives a normal binding curve which is hyperbolic in shape
-
Hemoglobin shows sigmoidal cooperative binding of oxygen
- direct result of its more complex subunit structure
-
P50: partial pressure of oxygen yielding 50% saturation of binding
- analogous to Km for the binding of substrates to enzymes
- direct result of its more complex subunit structure
- analogous to Km for the binding of substrates to enzymes
What kind of cooperativity does hemoglobin exhibit for oxygen?
Sequential cooperativity for oxygen binding:
- binding of oxygen to one subunit induces a conformational change that is partially transmitted to adjacent subunits
- transmission of the partial conformational change induces an increased affinity for oxygen by these adjacent subunits
- R=relaxed=high affinity; T=taut=low affinity
