13 - Heme Synthesis and Review of Hemoglobin Flashcards
What is the most common nutrient deficiency in the world?
Iron deficiency!
- Iron is an essential trace element that we get solely from the diet after birth
- Adult body iron content is 3-4 g
- Iron is conserved in the body (recyced) with only few mg’s lost daily from sloughing of intestinal cells or menstrual flow
Where is iron found in the body? How is it taken up?
The plama iron pool is iron-loaded transferrin (TF)
Iron-TF us taken up by cells via transferrin receptor.
Most of the body iron (1-2g) is found on Hb (heme) of RBC.
What are the functions of heme-containing porteins (there’s 3)?
- Transport oxygen: hemoglobin and myoglobin
- Electron transport: respiratory cytochromes
- Oxidation-reduction reactions: cytochrome P450 enzymes, catalase, NO synthase
What are the two major sites of heme synthesis?
Bone marrow (erythroblasts/reticulocytes)
Liver (hepatocytes)
How much of heme synthesis occurs in the bome marrow vs the liver?
Bone marrow: 80% of total synthesis; helps to make hemoglobin (6-7g Hb synthesized each day to replace heme loss through normal RBC turnover)
Liver: 20% of total synthesis ; helps make cytochrome P450 enzymes for drug detox
However, heme is also required for other important cellular proteins and is synthesized in virtually all cells, except matrue erythrocytes which lack mito.
What are porphyrins? Where are they found?
Cyclic tetrapyyroles capable of chelating to various metals to form essentual prosthetic groups for biological molecules. They emit right light when excited (~400-410 nm)
Heme is composed of a porphyrin derivative + a single ferrous ion (Fe2+). Heme is predominantly a planar molecule.
Heme is the _____ chelate of protoporphyrin ____.
Ferrous (Fe2+)
protoporphyrin IX.
Ferroprotoporphyrun IX (heme) is rapidly autooxidized to _______________ _____.
Ferriprotoporphyrin IX = “hemin”
Hemin contains ferrin Fe3+ iron.
There are 7 steps to heme synthesis. Which steps occur in the cytosol and which steps occur in the mitochondria?
The 1st step and last 3 steps occur in the mitochondria.
The intermediate steps occur in the cytosol.
What is the first step in heme synthesis? What catalyzes this step?
The condensation of glycine and succinyl-coA (intermediate of the TCA cycle), with decarboxylation, to yield 5-aminolevulinate (ALA)
5-aminolevulinate synthase (ALAS) catalyzes this commited step of heme synthesis.
Where is the enzyme ALAS, needed for the first step of heme synthesis, found in the cell? What does this enzyme depend on?
Localized to the inner mito membrane, but is encoded by a nuclear gene family and therefore the nascent protein must be imported into the mitochondrion.
ALAS is a pyridoxal phosphate (PLP) dependent enzyme.
What are the two forms of ALAS (enzyme in the first step of heme synthesis)?
ALAS1 is the liver isoform that’s ubiquitously expressed and present in most tissues.
ALAS2 is the erythroid/reticulocyt-specific isoform.
How is the ALAS1 isoform regulated in the liver?
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 mitochondrial import.
What can alter the activity of ALAS1 in the liver?
~100 drugs/metabolites can increase ALAS1 activity (via increased transcription)
Many drugs are metabolized by P450s in the liver; many drugs increase the synthesis of P450 enzymes, thereby increasing the demand for heme.
How is ALAS2 in reticulocytes regulated?
Heme biosynthesis in erythroid cells is NOT regulated by feedback repression of ALAS2 by heme.
In reticulocytes, heme stimulates synthesis of globin and ensures that heme and globin are synthesized in the correct ratio for assembly into Hb.
What is translation of ALAS2 mRNA dependent upon?
Translation of ALAS2 mRNA depends on iron availability.
5’UTR of ALAS2 mRNA has “iron sensors”: stem-loop iron responsive elements (IREs) similar to that found in ferritin and transferrin receptor mRNAs.
- Low iron = repression of ALAS3 translation; high iron allows translation initiation
Drugs that cause a marked elevation in ALAS1 activitym, such as phenobarbital, have what effect on ALAS2?
They have no effect on ALAS2.
What is the second stap in heme synthesis? What catalyzes this step?
ALA dehydrogenase (ALAD) is a cytosolic enzyme that catalyzes the condensation of two molecules of ALA to form on molecule of porphobilinogen (PBG) (this is the first pathway intermediate that inclued a pyrrole ring.
This enzyme requires Zn2+ which is complexed to cysteine.
What can interfere with the activity of ALA dehydrogenase (ALAD) (enzyme in the second step of heme synthesis)?
This enzyme requires Zn2+.
Lead and other heavy metals can displace Zn2+ and eliminate catalytic activity.
- Lead poisening results in an increase of ALA in the urine and blood and can cause clinical manifestations that mimic acute porphyrias.
How is lead absorbed in the body? In what instances are people exposed to lead?
In the lungs via inhalation, intestine by ingestion, and skin by absorption.
- Occupational exposure (gasoline, soldering, lead-based paints, storage batteries) are most common causes in adults, wish respiratory entry being the major route.
- Absorption through gut major route in children (lead pipes contaminating water)
How does lead affect the body?
It impacts virtually every system in the body:
- it inhibits enzymes/proteins by substituting for calcium or zinc
- best understood toxic effects involve heme synthesis
In what three places does lead accumulate in the body? What is it’s half life? Where does most of the absorbed lead go?
Accumulates in blood, soft tissue, and bone.
Lead in serum half life is ~30 days, although excreted in urine, short half-life in serum is due to distribution to the tissues.
Lead is incorporated into bone matrix as a substitute for hydrozxyapetite; bone functions as a long-term storage pool for 90-95% of absorbed lead.
Bone lead is regarded as 2 distinct pools. What are they?
Inert pool: with half life of decades
Labile pool: readily exchanges/mobilizes to serum and soft tissues
What are the serum levels of lead toxicity in adults and children? What is the time of onset of symptoms?
Adults: >40 microg/dL
Children: >25 microg/dL, although cognitive impairment documented at <10 microg/dL
- lead is able to cross the placenta to reach the fetus
Time of onset of symtoms is 2 weeks to ~30 years.
What effect does lead have on ALA dehydratase (aka porphobilinogen synthase)? What impact does this have on the body?
It inhibits it, resulting in elevated urine/blood ALA. Impaired heme synthesis leads to de-repression of transcription of ALAS gene.
High ALA is through to cause some of the neurological effects of lead poisening, although Pb2+ may directly affect the nervous system too.
What is the third step of hemoglobin synthesis? What enzyme catalyzed this step? (I’m sorry, there was no good way to make this card shorter :( )
Porphobilinogen deaminase (PBGD) catalyzes the head-to-tail condensation of four porphobilinogen molecules to form a linear tetrapyrrole (hydroxymethylbilane)
This tetrapyrrole can spontaneously cyclize to form uroporphyrinogen 1 which contains a symmetric arranagement of side chains and is NOT in the normal pathway for heme biosyn.
However! PBGD is tightly associated with an enzyme uroporphyrinogen cosynthase (UROS) that directs the stereochemistry of the condensation reaction to yield uroporphyrinogen III isomer which IS in the pathway for heme biosyn.
What is the 4th step of heme biosynthesis?
Uropophyrinogen decarboxylase (UROB) catalyzes the decarboxylation of acetate side chains to methyl groups on uroporphyrinogens III to yeild coprophorphyrinogen III.
What is the 5th step of heme biosynthesis? What catalyzes this step and where is this enzyme located?
Coproporphyrinogen III is transported to the intermembrance space where it’s converted to protoporphyrinogen IX by coproporphyrinogen III oxidase (CPO). This oxidase converts specific propionic acid side chains to vinyl groups.
CPO is located in the intermembrane space in the mito, which means it’s product (protoporphyrin IX) must cross the inner mito membrane because heme is formed within the inner membrane.
What is the 6th step of heme biosynthesis? What catalyzes this reaction?
Another mitochondrial oxidase, protoporphyrinogen IX oxidase (PPO) converts protoporphyrinogen IX to protopohphyrin IX (moving double bonds).
What is the 7th step in heme biosynthesis? What enhances the rate of this reaction?
Insertion of Fe2+ into protoporphyrin IX generates HEME and occurs spontaneously at a slow rate. This rate is enhanced by ferrochelatase.
What are the result of early and late defects in the heme biosynthetic pathways (porphyrias)?
Early defects: causes accumulation of ALA, porphobilinogen that causes neurologic dysfunction.
Later defects: accumulation of tetrapyrroles, but NOT porphobilinogen. Results in sunlight-induced cutaneous lesion. In the presence of oxygen, UR irradiation of cyclic tetrapyrroles generates reactive ocygen species that can cause cellular damage.
What form of iron is capable of binding O2?
Fe2+ is the ferrous form of iron that binds O2.
(Fe3+ is the ferric form of iron that CANNOT bing O2 and is present in the inactive form of Hb called methemoglobin (metHb).
How does the binding curve of hemoglobin compare to that of myoglobin? How does this relate to their function?
Myoglobin has a normal binding curve that’s hyperbolic. This is consistent with myoglobins function of storage.
Hemoglobin shows sigmoidal cooperative binding of oxygen that is a direct result of it’s more complex subunit structure.
The cooperative binding of oxygen by Hb is critical for it’s efficiency in loading oxygen in the lungs and unloading it in the peripheral tissues.
Hemoglobin exhibits cooperativity for oxygen binding. What does this mean?
Binding of oxygen to one subunit induces a conformational change that’s partially transmitted to adjacent subunits.
The transmission of the partial conformational change induces an increased affinity for oxygen by these subunits.
R is the relaxed state with high affinity for oxygen; T is the taut state with low affinity for oxygen.
What effect does carbon monoxide have on hemoglobin?
CO has a 250-fold higher affinity for Hb than oxygen does. When bound to the heme group of one subunit, it causes all four subunits to “lock: in the R conformation (relaxed, high affinity), thereby limiting oxygen release in peripheral tissues.
How does O2 binding chane the conformation of a Hb subunit?
Without O2 bound, the heme Fe2+ is pulled away from the plane of the porphyrin ring by a His residue.
When O2 binds, it pulls the Fe2+ back into the plane of the ring, and that moves the His residue and its whole section of the polypeptide chain.
That in turn causes the Hb sunits to shift relative to one another in an arrangement that favors the R (relaxed/high affinity) state.
What molecules allosterically regulate O2 binding to hemoglobin?
What effect does 2,3 DPG have on Hb states? When do DPG levels increase?
It stabilizes the T-state, making it easier for Hb to release oxygen.
DPG levels increase in response to high altitudes. This increase in DPG concentration allows RBCs to adapt to hypoxia by making it easier for O2 to dissociate from Hb.
Changes of DPG occur hours to days, which is why it takes a few days to adjust to high altitudes.
How do pH, DPG, and temperature impact the hemoglobin associated/dissociation curve?
How does fetal hemoglobin differ from adult Hb?
Developmental forms of Hb differ not only in subunits, but also in their binding affinity for O2.
The higher affinity of fetal Hb (HbF; alpha2,gamma2) means teh fetus’ circulation can draw ) O2 from maternal blood at the pO2 present in placenta.
What causes sickle cell anemia?
Homozygous recessive disease caused by a point mutation in adult B-globin gene that causes substitution of valine (Val) for glutamic acid (Glu) at aa 6.
This valine is hydrophobic and its presence creates a sticky patch on deoxyHb that leads to polymerization of Hb tetramers into long chains. Those intracellular fibers cause the sickle shape and reduced deformablity of RBCs and that leads to problems in their passage through microcirculation. This is the basis for vaso-occlusive problems seen in sickle cell.
What is B thalassemia major vs minor? How does the anemia associated with each differ?
