Download Synthesis of Heme

Molecular Biochemistry II
Synthesis of Heme
Copyright © 1999-2008 by Joyce J. Diwan.
All rights reserved.
CH3
CH3
S
HC
CH2 protein
N
H3C
CH3
N

OOC
CH2 CH2
Fe
N
CH
N
S
CH2
protein
CH3
CH2
CH3
CH2
COO
Heme c
Heme is the prosthetic group of hemoglobin, myoglobin, &
cytochromes. Heme is an asymmetric molecule. E.g., note the
positions of methyl side chains around the ring system.
The heme ring system is synthesized from glycine &
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 enters
Krebs Cycle as acetyl-CoA, and the labeled carbon
becomes incorporated into succinyl-CoA, the more
immediate precursor of heme.
O

OOC
CH2
CH2
C
S-CoA +
succinyl-CoA
d-Aminolevulinic
Acid Synthase

OOC
CH2
NH3+
glycine
H+
CoA-SH
CO2
O

OOC
CH2
CH2
C
CH2
NH3+
d-aminolevulinate (ALA)
Heme synthesis begins with condensation of
glycine & succinyl-CoA, with decarboxylation,
to form d-aminolevulinic acid (ALA).
H
Pyridoxal phosphate
(PLP) serves as coenzyme
for d-Aminolevulinate
Synthase (ALA Synthase),
an enzyme evolutionarily
related to transaminases.
Condensation with
succinyl-CoA takes place
while the amino group of
glycine is in Schiff base
linkage to the PLP aldehyde.
CoA & the glycine carboxyl
are lost following the
condensation.
O
O
P
O
C
H2
C
OH
O
O

N
CH3
H
Pyridoxal phosphate (PLP)
H2C
COO
N+
HC
O
O
H2
C
P
O
H
O
O

N
H
CH3
glycine-PLP Schiff base (aldimine)
ALA Synthase is the committed step of the heme
synthesis pathway, & is usually rate-limiting for the
overall pathway.
Regulation occurs through control of gene transcription.
Heme functions as a feedback inhibitor, repressing
transcription of the ALA Synthase gene in most cells.
A variant of ALA Synthase expressed only in developing
erythrocytes is regulated instead by availability of iron in
the form of iron-sulfur clusters.
COO
COO
CH2
CH2
CH2
CH2
C
+
O
C
PBG Synthase
2 H2O
O
CH2
CH2
NH3+
NH3+
2 d-aminolevulinate
(ALA)
H2C
COO
COO
CH2
CH2
CH2
C
C
C
CH
NH3+
N
H
porphobilinogen
(PBG)
PBG Synthase (Porphobilinogen Synthase), also
called ALA Dehydratase, catalyzes condensation of
two molecules of d-aminolevulinate to form the
pyrrole ring of porphobilinogen (PBG).
The reaction
mechanism involves
2 Lys residues & a
bound cation at the
active site.
Cation in mammalian
enzyme is Zn++.
HOOC
CH2
H2C
CH2
LysEnzyme
CH2 CH2
C
H2C
NH2
NH+
ALA in Schiff base linkage
to active site lysine
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.
COO
HOOC
CH2
H2C
CH2
LysEnzyme
CH2 CH2
C
H2C
NH2
COO
CH2
CH2
CH2
NH+
ALA in Schiff base linkage
to active site lysine
A proposed reaction mechanism
is based on crystal structures of:
H2C
NH3
+
N
H
Porphobilinogen (PBG)
 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.
The Zn++ binding sites 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, as impaired heme synthesis leads to
de-repression of transcription of the ALA Synthase gene.
High ALA is thought to cause some of the neurological
effects of lead poisoning, although Pb++
COO
COO
also may directly affect the nervous system.
ALA is toxic to the brain, perhaps due to:
• Similar ALA & neurotransmitter GABA
(g-aminobutyric acid) structures.
• ALA autoxidation generates reactive
oxygen species (oxygen radicals).
CH2
CH2
CH2
CH2
C
O
CH2
CH2
NH3+
NH3+
ALA
GABA
COO
N
H
pyrrole
Porphobilinogen
(PBG) is the first
pathway intermediate
that includes a
pyrrole ring.
COO
CH2
CH2
CH2
H2C
NH3
+
N
H
Porphobilinogen (PBG)
The porphyrin ring is formed by condensation of 4
molecules of porphobilinogen.
Porphobilinogen Deaminase catalyzes successive PBG
condensations, initiated in each case by elimination of
the amino group.
COO-
Enz
S
COO
COO-
CH2
COO-
CH2
CH2
CH2
CH2
CH2
N
H
dipyrromethane
-
N
H
PBG Deaminase
PDB 1PDA
Porphobilinogen Deaminase has a dipyrromethane
prosthetic group, linked at the active site via a cysteine S.
The enzyme itself catalyzes formation of this prosthetic
group.
COOCOO-
CH2
COO- COO-CH
COO-
Enz
S
COO- CH2
COO- CH2
CH2
CH2
N
H
CH2
CH2
2
CH2
CH2
CH2
N
H
NH
HN
NH
HN
CH2 COO-
CH2 COOCH2
CH2 CH2
CH2
COO-COO-
CH2
COO-
PBG units are added to the dipyrromethane until a linear
hexapyrrole has been formed.
COO-
Porphobilinogen
Deaminase is
organized in 3
domains.
Predicted
interdomain
flexibility may
accommodate
the growing
polypyrrole in the
active site cleft.
-
OOC
CH2
COO-
CH2
CH2
CH2
NH
HN
NH
HN
CH2
CH2 COO-
CH2
COO-
HO
CH2
CH2 CH2
CH2
COO-COO-
CH2
hydroxymethylbilane
COO-
Hydrolysis of the link to the enzyme's dipyrromethane
releases the tetrapyrrole hydroxymethylbilane.
COO-
hydroxymethylbilane
-
OOC
COO-
uroporphyrinogen
III
-
CH2
COO-
CH2
COO
CH2
CH2
CH2
CH2
CH2
CH2
NH
HN
NH
HN
CH2 COO-
-
OOC CH2
NH
HN
NH
HN
CH2
CH2 COO-
CH2
COO-
HO
C
C
CH2
C
COO-
-
OOC CH2
C
CH2
CH2 CH2
CH2
COO-COO-
CH2
COO-
Uroporphyrinogen III
Synthase
CH2
CH2
CH2
CH2
COO-
COO-
Uroporphyrinogen III Synthase converts the linear
tetrapyrrole hydroxymethylbilane to the macrocyclic
uroporphyrinogen III.
Uroporphyrinogen
III Synthase
catalyzes ring
closure & flipping
over of one pyrrole
to yield an
asymmetric
tetrapyrrole.
This rearrangement
is thought to
proceed via a spiro
intermediate.
COO-
-
OOC
CH2
COO-
CH2
CH2
CH2
CH2
N
NH
HN
C
HN
CH2
HC
CH2
COO-
COO-
CH2
CH2
COOCH2
CH2
COO-
COO-
CH2
postulated
intermediate
COO-
hydroxymethylbilane
-
OOC
COO-
uroporphyrinogen
III
-
CH2
COO-
CH2
COO
CH2
CH2
CH2
CH2
CH2
CH2
NH
HN
NH
HN
CH2 COO-
-
OOC CH2
NH
HN
NH
HN
CH2
CH2 COO-
CH2
COO-
HO
C
C
CH2
C
-
COO
-
OOC CH2
C
CH2
CH2 CH2
CH2
COO-COO-
CH2
COO-
Uroporphyrinogen III
Synthase
CH2
CH2
CH2
CH2
COO-
COO-
Note the distribution of acetyl & propionyl side chains,
as flipping over of one pyrrole yields an asymmetric
tetrapyrrole.
The active site of
Uroporphyrinogen III
Synthase is 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
PDB 1JR2
Synthase
Uroporphyrinogen III is the precursor for synthesis of
vitamin B12, chlorophyll, and heme, in organisms that
produce these compounds.
Conversion of uroporphyrinogen III to protoporphyrin IX
occurs in several steps.
-
COO
-
protoporphyrin IX
uroporphyrinogen III
CH2
COO-
CH2
CH2
CH2
CH
OOC CH2
CH2
-
CH2 COO
CH3
CH CH2
H3C
NH
HN
NH
NH
HN
N
-
CH2
OOC CH2
COO-
N
HN
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).
CH2
protoporphyrin IX
CH2
CH
CH3
CH
CH CH2
H3C
NH
N
N
Fe++
heme
CH3
CH CH2
H3C
Fe
HN
N
H3C
CH3
CH2
CH2
CH2
COO-
N
N
2H+
N
H3C
Ferrochelatase
CH3
CH2
CH2
CH2
CH2
CH2
COO-
COO-
COO-
Fe++ is added to protoporphyrin IX via Ferrocheletase, a
homodimeric enzyme containing 2 iron-sulfur clusters.
A conserved active site His, along with a chain of anionic
residues, may conduct released protons away, as Fe++ binds
from the other side of the porphyrin ring, to yield heme.
Regulation of transcription or post-translational processing
of enzymes of the heme synthesis pathways differs between
erythrocyte 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 one
of 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.
Occasional episodes of severe neurological symptoms are
associated with some porphyrias.
 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, 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.
Question: How do you think episodes of acute neurological
symptoms 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 repress
transcription of the gene for ALA Synthase, rate-limiting
for the pathway and the source of excess ALA.
View an amination of the heme synthesis pathway.
Regulation of iron absorption & transport.
Iron for synthesis of heme, Fe-S centers & other
non-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.
transferrin with
bound Fe
extracellular space
transferrin
receptor
receptor-mediated
endocytosis
Iron is transported in blood serum bound to the protein
transferrin.
The plasma membrane transferrin receptor mediates
uptake of the complex of iron with transferrin by cells
via receptor mediated endocytosis.
Iron is stored within cells as a complex with the protein
ferritin.
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 iron
levels involves regulation of ferroportin expression.
 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.
Hereditary hemochromatosis is a family of genetic
diseases 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 to
unrestrained ferroportin activity, with high dietary intake
and high % saturation of serum transferrin with iron.
Organs particularly affected by accumulation of excess
iron include liver and heart.