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Introduction to Porphyrins
Haem is a metalloporphyrin. It has four pyrrole rings that are linked to each other via methenyl bridges. Then four nitrogens of the pyrrole ring bind ferrous iron in haem. If ferric iron is bound, then hemin is formed. This sqadia lecture focuses on Synthesis of Haem. Porphyrins are introduced as complex structures consisting of 4 pyrrole rings, united by “methyne” bridges (or methylidene bridges). They form the prosthetic groups(Fe-porphyrins) of conjugated proteins, viz. Haemoglobin of mammalian erythrocytes, Myoglobin of muscle. Porphyrins are synthesised partly in the mitochondrion and partly in cytosol of aerobic cells. There are 3 Stages of biosynthesis. Stage I: Synthesis of δ-Amino Levulinic acid occurs in mitochondria. Biosynthesis begins with the condensation of ‘succinyl CoA’ and glycine to form ‘α-amino-β-Ketoadipic acid”. α-amino-β-ketoadipic acid then undergoes decarboxylation to produce δ-ALA. Stage II: Synthesis of coproporphyrinogen III & I which occurs in cytosol, and Stage III: Synthesis of protoporphyrin IX, which occurs in mitochondria again. δ-ALA synthetase enzyme is very unstable, low in concentration in tissues, and the main rate-limiting enzyme in the synthetic pathway. Many erythropoietic substances including hormones stimulate haem synthesis by inducing the production of the enzyme. End product ‘haem’ inhibits the enzyme by “feedback” inhibition. Haem also causes a repression of the synthesis of the enzyme, “end-product repression”.
Stage II: Synthesis of Coproporphyrinogen III and I
δ -ALA comes out of mitochondrion into the cytosol. Two molecules of δ-ALA condense further to form a molecule of porphobilinogen, which is the precursor of ‘pyrrole’ ring. The reaction is catalyzed by the enzyme δ-ALA dehydratase, for which Cu++ is required as a cofactor. This is a second rate-limiting enzyme. In presence of a porphobilinogen deaminase, 4 mol. of porphobilinogens condense, losing 4 mol. of NH3 and forms uroporphyrinogen I, in which the acetic acid and propionic acid side chains alternate. “Dipyrroles” and “tetrapyrroles” may be formed as intermediates. Concomitant operation of an isomerase (also called as uroporphyrinogen III cosynthetase) with deaminase, results in reversal of one porphobilinogen residue, so that the cyclisation results in the formation of uroporphyrinogen III. In this, in IV pyrrole ring, acetic acid and propionic acid side chains are “reversed”. Formation of coproporphyrinogen I and III is catalyzed by uroporphyrinogen decarboxylase of the four acetic acid side chains of the corresponding uroporphyrinogens to “methyl groups” results in coproporphyrinogens I and III. In the formation of Protoporphyrin IX, coproporphyrinogen III enters mitochondrion. An oxidative decarboxylase system containing flavins as coenzyme converts coproporphyrinogen III to protoporphyrinogen IX. Protoporphyrinogen IX is converted to protoporphyrin IX by another oxidase enzyme. Insertion of an atom of Fe++ into central position of protoporphyrin IX is catalyzed by haem synthetase (ferrochelatase). The “haem” which is produced is then coupled to various proteins and thus form the conjugated proteins, viz. haemoglobin, myoglobin, cytochrome C, catalases and peroxidases.
Regulatory Influences and Effects of Inhibitors
Effect of O2 on haem synthesis is rather complex. In vivo: stimulated by low O2 tension (e.g. living at high attitudes). In vitro: conversion of porphobilinogen to uroporphyrinogen and protoporphyrin to haem, are both inhibited by O2. Enzymes which catalyse the synthesis and utilisation of δ-ALA are important sites of regulation. Haem, the end-product of the metabolic sequence, inhibits the activity of synthetase. Many compounds of diverse structures, viz. certain insecticides, carcinogens and others, when administered to human beings can result in marked increase in hepatic δ-ALA synthetase, leading to increased porphyrins. Lead is known to produce profound abnormalities in porphyrin metabolism. It inhibits δ-ALA synthetase, δ-ALA dehydratase and haem synthetase ‘in vitro’. Glucose can prevent induction of δ-ALA. Hypoxia increases δ -ALA synthetase activity in erythropoietic tissues. Administration of haematin in vivo can prevent the drug-mediated ‘derepression’ of δ -ALA synthetase.
Synthesis of Hemoglobin
In adult man, the synthesis of Hb is restricted normally to the immature red cells of bone marrow. Three components are required: Protoporphyrin IX: synthesized as described earlier. Globin: produced by the usual mechanisms of protein synthesis. Iron: sources, absorption, storage, etc. In addition to the actual constituents of haemoglobin, certain vitamins and other factors are also required such as Pantothenic acid: It is necessary for synthesis of CoASH, required for the substrate succinyl-CoA. Pyridoxal-P: It is required as a coenzyme for the activity of δ -ALA synthetase. Vit C (ascorbic acid): It is essential as it helps in absorption of Fe from the gut, converts Fe+++ to Fe++ and it helps in mobilization of Fe from ferritin. Intrinsic factor: It is necessary for vit B12 absorption. Vit B12 and Folic Acid is required indirectly as the rapid rate of cell growth and division occurring during erythropoiesis results in a correspondingly rapid rate of nucleic acid synthesis, which require B12 and folate, to ensure adequate supplies of formate for purine synthesis. Synthesis of Hb appears to proceed concurrently with the maturation of erythrocytes. The primitive red cells contain free porphyrins rather than Hb. As the red blood cells mature, the content of free porphyrin decreases and that of Hb rises. Haem has been shown to stimulate the synthesis of ‘globin’ on the ribosomal level.
Porphyrias and Porphyrinurias Clinical Aspect
When the blood levels of coproporphyrins and uroporphyrins are increased above normal level and excreted in urine/faeces, the condition is called porphyria. Under these terms are included a number of syndromes, some are hereditary and familial, and some others are acquired. Several different classifications of porphyrias have been proposed. Hereditary porphyrias are divided into two main groups based on the porphyrin and porphyrin precursors content in bone marrow (erythropoietic) and in liver (hepatic). Hepatic porphyrias may further be subdivided into 3 groups depending on the clinical presentation and enzyme deficiency. Hereditary Porphyrias are congenital erythropoietic porphyrias. These are rare inherited disorder. Affected individuals exhibit abnormal sensitivity to light (photosensitivity) and develop skin lesions. In Hepatic Porphyrias, organ responsible for the dysfunction is the liver, in which there occurs abnormal and excessive production of porphyrins (chiefly type III), their precursors δ-ALA and porphobilinogen. Porphyria Cutanea Tarda is the most common form, seen in South African whites. It is characterized principally by skin photosensitivity. Normally only small amounts of coproporphyrins of type I and III, 60 to 280 μg in the ratio of 70 per cent type I and 30 per cent type III are excreted in urine per day in normal health. Uroporphyrins are excreted only in negligible amounts of 15 to 30 μg per day. The faeces normally contain 300 to 1000 μg of coproporphyrins per day, mostly of the type-I. When porphyrins are dissolved in strong mineral acids or in organic solvents and illuminated by UV light, they emit a strong red fluorescence. An interesting application of the photodynamic properties of porphyrins is their possible use in the treatment of certain types of cancer, a procedure called cancer phototherapy.