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). The extensive conjugated system makes the compound chromatic, hence the name porphyrin, from a Greek word for purple. The macrocycle has 22 pi electrons, 18 of which are active in the conjugated system. The names of the porphyrins of interest consist of a word and a number, e.g., uroporphyrin III. The word denotes the kinds of constituents found on the ring, and the number denotes how they are arranged. 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.

Porphyrinogens are more reduced than the corresponding porphyrins, so oxidation of a porphyrinogen produces the corresponding porphyrin.

Heme Synthesis
Site: Partly in the mitochondria and partly in the cytoplasm. Process begins in the mitochondria because one of the precursors is found only there. Since this reaction is regulated in part by the concentration of heme, the final step (which produces the heme) is also mitochondrial. 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 deltaaminolevulinate.

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 5beta 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 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 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.

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.






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