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Porphyrins

¾ A porphyrin is a heterocyclic macrocycle made from 4 pyrrole


subunits linked on opposite sides (α position) through 4 methine
bridges (=CH-). The macrocycle, therefore, is more aromatic than
the related corrins, chlorins (2,3-dihydroporphyrin) and
bacteriochlorophylls (2,3,12,13-tetrahydroporphyrin).
¾ The extensive conjugated system makes the compound chromatic,
hence the name porphyrin, from a Greek word for purple.
¾ The macrocycle has 22 pi electrons, 18 of which are active in the
conjugated system.
¾ The names of the porphyrins of interest consist of a word and a
number, e.g., uroporphyrin III.
¾ The word denotes the kinds of constituents found on the ring, and
the number denotes how they are arranged.
¾ Iron can be added to protoporphyrin IX. The oxidation state of the
iron ion and the identity of the negatively charged counterion
determine the name of the product.

If iron (II), or ferrous iron, is added the product is heme.


If iron (III), or ferric iron, is added the product is

• Hemin if the counterion is chloride.


• Hematin if the counterion is hydroxide.

¾ Porphyrinogens are more reduced than the corresponding


porphyrins, so oxidation of a porphyrinogen produces the
corresponding porphyrin.

Heme Synthesis
¾ Site: Partly in the mitochondria and partly in the cytoplasm.
¾ Process begins in the mitochondria because one of the precursors
is found only there.
¾ Since this reaction is regulated in part by the concentration of
heme, the final step (which produces the heme) is also
mitochondrial.
¾ Many of the intermediate steps are cytoplasmic. Notice in the
diagram of the pathway that there is a branch with no apparent
useful end product.
Delta-aminolevulinic acid synthase (ALA synthase)
The delta-aminolevulinic acid synthase (ALA synthase) reaction occurs in
the mitochondria.

The substrates are

• succinyl CoA (from the tricarboxylic acid cycle)


• glycine (from the general amino acid pool)

An essential cofactor is pyridoxal phosphate (vitamin B-6).

• The reaction is sensitive to nutritional deficiency of this vitamin.


• Drugs which are antagonistic to pyridoxal phosphate will inhibit it.
Such drugs include
o penicillamine
o isoniazid (isonicotinic acid hydrazide)

The reaction occurs in two steps.

• Condensation of succinyl CoA and glycine to form enzyme-bound


alpha-amino-beta-ketoadipate.
• Decarboxylation of alpha-amino-beta-ketoadipate to form delta-
aminolevulinate.
This is the rate-limiting reaction of heme synthesis in all tissues, and it
is therefore tightly regulated.

There are two major means of regulating the activity of the


enzyme.

The first is by regulating the synthesis of the enzyme protein. This is


important because its half life is only about one hour. Enzyme synthesis
is repressed by heme and hematin. It is stimulated by

• Barbiturates (as a result, these drugs exacerbate certain


porphyrias).
• Steroids with a 4, 5 double bond, such as testosterone and
certain oral contraceptives. This double bond can be reduced
by two different reductases to form either a 5-alpha or a 5-
beta product. Only the 5-beta product affects synthesis of
ALA synthase. Since the 5-beta reductase appears at
puberty, some porphyrias are not manifested until this age.

The second control is feedback inhibition by heme and hematin,


presumably by an allosterica mechanism.

Hence, heme has a dual role in decreasing its own rate of synthesis.

The product of the reaction, ALA, diffuses into the cytoplasm, where the
next several steps of heme synthesis occur.
ALA dehydratase reaction
The ALA dehydratase reaction occurs in the cytoplasm; the product is
porphobilinogen

The substrates are two molecules of ALA.

The reaction is a condensation to form porphobilinogen, the


first pyrrole.
Two molecules of water are released. The asymmetry of the reaction
relative to the two molecules of substrate results in the pyrrole ring
having two different substituent groups:

• acetic acid
• propionic acid.

ALA dehydratase is a -SH containing enzyme.

• It is very susceptible to inhibition by heavy metals, especially lead.


• increased urinary excretion of its substrate is a good indicator of
lead poisoning
Uroporphyrinogen I synthase & uroporphyrinogen
III cosynthase

Production of uroporphyrin III requires two enzymes.

The substrates are four molecules of porphobilinogen.

The first reaction is catalyzed by uroporphyrinogen I synthase.

• The porphobilinogen molecules lose their amino groups.


• A linear tetrapyrrole with alternating acetic acid and propionic acid
groups is produced. This linear molecule cyclizes slowly
(nonenzymatically) to yield uroporphyrinogen I. Without the second
reaction (below), the heme synthesis pathway would end with
porphyrinogens of the I series, which have no known function.

The second reaction is catalyzed by uroporphyrinogen III cosynthase.


This enzyme rapidly converts the alternating linear tetrapyrrole to the
cyclic uroporphyrinogen III, which has the constituents of its IV ring
reversed: AP AP AP PA. This is the physiologically useful product.
Uroporphyrinogen decarboxylase
Uroporphyrinogen decarboxylase decarboxylates the acetic acid groups,
converting them to methyl groups.

• The physiologically significant substrate is uroporphyrinogen III.


• The product is coproporphyrinogen III, which is transported back
to the mitochondria, where the remainder of heme synthesis
occurs.
• The constituent pattern in the coproporphyrinogen is MP MP MP
PM.
• Uroporphyrinogen decarboxylase also acts on uroporphyrinogen I,
yielding coproporphyrinogen I. Coproporphyrinogen I has no
known function, and its formation is thought to be a blind
pathway.
Coproporphyrinogen III oxidase
The mitochondrial enzyme, coproporphyrinogen III oxidase, catalyzes the
next reaction.

• The substrate is coproporphyrinogen III.


• The reaction is conversion of the propionic acid groups of rings I
and III to vinyl groups. We now have the final substituent pattern
of MV MV MP PM (note that "Petrarchan" pattern of the last four
substituent groups).
• The product is protoporphyrinogen IX. (Some naming systems
would call this protoporphyrinogen III to preserve the logic of the
nomenclature, but "protoporphyrinogen III" is a departure from a
time-honored tradition of referring to this and subsequent
compounds by the number "IX.").
Protoporphyrinogen IX oxidase
Protoporphyrinogen IX oxidase converts the methylene bridges between
the pyrrole rings to methenyl bridges. Resonance of double bonds around
the entire great ring, with its resulting stabilization, is now possible.

Ferrochelatase
Ferrochelatase adds iron (II) to protoporphyrin IX, forming heme.

• The enzyme requires iron (II), ascorbic acid and cysteine (reducing
agents).
• Ferrochelatase is inhibited by lead.
Physiological regulation of heme synthesis
Substrate availability: iron (II) must be available for ferrochelatase.

Feedback regulation: heme is a feedback inhibitor of ALA synthase.

Subcellular localization: ALA synthase is in the mitochondria, where


the substrate, succinyl CoA, is produced. ALA synthase is synthesized in
the cytoplasm, and is transported into the mitochondria (like many other
mitochondrial proteins). Its transport across the mitochondrial
membrane may be regulated.

In erythropoietic cells, heme synthesis is coordinated with globin


synthesis. If heme is available, globin synthesis proceeds. If heme is
absent:

• A cAMP independent protein kinase is active.


• The kinase phosphorylates and thereby inactivates, the eukaryotic
initiation factor, eIF-2. This prevents further globin synthesis

Effects of drugs and steroids: Remember, certain drugs and steroids


can increase heme synthesis via increased production of the rate-limiting
enzyme, ALA synthase.

Porphyrias
Porphyrias may be divided into two major types.
Erythropoietic porphyria is a defect of porphyrin metabolism of
blood-producing tissues.
Hepatic porphyria is a defect in porphyrin metabolism of the liver.
Either type may be hereditary (caused by a gene defect) or acquired
(due to poisoning).

The symptoms of porphyria cannot be completely explained by


our current understanding of the biochemical defect.
In all cases there is an identifiable abnormality of the enzymes
which synthesize heme.
This leads to accumulation of intermediates of the pathway and a
deficiency of heme.
Accumulation of porphyrinogens in the skin can lead to
photosensitivity.
The neurological symptoms cannot be explained. They are
especially puzzling since the metabolic defect is confined to non-
neural tissue, and no known diffusible intermediate can cause the
observed neurological symptoms.

Examples of Porphyria
Two of the several types of porphyria will serve to illustrate some of the
biochemical issues involved.

Acute intermittent porphyria is a defect of hepatic


uroporphyrinogen I synthase activity.

• Porphobilinogen (the substrate) accumulates, and is excreted in


the urine. Note: analysis of the urine for porphobilinogen is
relatively simple.
• Heme synthesis is reduced. As a result, there is no feedback
inhibition of ALA synthase, nor is there repression of synthesis of
the enzyme protein. ALA synthase activity therefore increases.
• Insufficient 5-alpha steroid reductase activity shunts steroids into
the 5-beta reductase pathway, whose products further stimulate
ALA synthase.
• There are neurological symptoms, including agitation, which
cannot be explained. Administration of barbiturates to sedate an
agitated patient would be counterproductive, as it would further
activate ALA synthase, and result in more rapid clinical
deterioration.

Hereditary coproporphyria is a defect of hepatic


coproporphyrinogen oxidase.

• Large amounts of coproporphyrin III are excreted in the feces.


• Poorly understood neurological symptoms occur.
• Skin lesions may occur.
o Porphyrinogens are subject to light-catalyzed oxidation to
their corresponding porphyrins.
o Porphyrins absorb visible light strongly. Exposure to strong
visible light (sunlight) may cause pruritis, edema and so
forth. The most damaging wavelength is 405 nm, which is
the peak of the absorption spectra of porphyrins. This
wavelength is not blocked by window glass.

Two Steps of Heme Degradation


Heme is degraded in two steps to bilirubin, which is conjugated to
glucuronic acid and excreted.
The first reaction is cleavage of the heme ring by a microsomal
heme oxygenase.

The substrates for the reaction are

• heme
• three molecules of oxygen
• NADPH

The reaction is a cleavage of the ring between the I and II pyrrole rings.

• The alpha-methylene group is released.


• The product is symmetric with respect to the propionic acid
groups.

The products are

• biliverdin
• carbon monoxide (this is the only endogenous source of carbon
monoxide)
• iron (II)
• NADP+
In the second reaction biliverdin reductase reduces the central
methene bridge of biliverdin, producing bilirubin.

Conversion of biliverdin to bilirubin requires NADPH as a reducing agent.

Bilirubin
The high lipid solublity of bilirubin determines its behavior and its
further metabolism.

Its lipid solubility dictates

• that it must be transported in the blood by a carrier; the


physiological carrier is serum albumin.
• that it is soluble in the lipid bilayers of cell membranes.

Bilirubin must be conjugated to a water-soluble substance

This increased its water solubility, decreases its lipid solubility and eases
its excretion. Conjugation is accomplished by attaching two molecules of
glucuronic acid to it in a two step process.

The substrates are

• bilirubin (or bilirubin monoglucuronide)


• UDP-glucuronic acid.
The reaction is a transfer of two glucuronic acid groups
sequentially to the propionic acid groups of the bilirubin. The
major product is bilirubin diglucuronide.

Bilirubin diglucuronide is excreted in the bile. It is subject to subsequent


transformations to other species by the intestinal flora.

Further degradation occurs by the breakdown of bilirubin diglucuronide


to Urobilinogen.

This Urobilinogen is converted to Urobilin (Kidneys), and Stercobilin


(Microbial enzymes in intestine).

This Urobilin formed is excreted through urine and the Stercobilin is


excreted through the faeces.

The following reactions represent this process of excretion of bile


pigments.
Urobilinogen

Stercobilin

Urobilin Urine Faeces

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