Enzymes heme biosynthetic

Although not specific for kerosene, aminolevulinic acid (ALA) could potentially be used as an adjunct or supplemental biomarker for kerosene exposure. Kerosene may affect heme metabolism by decreasing the activities of enzymes in the heme biosynthetic pathway (hepatic -ALA dehydratase and -ALA synthetase) (Rao and Pandya 1980). Therefore, it may be possible that this effect would generate increased ALA in the urine of exposed individuals. Additional studies of acute, intermediate, and chronic exposure are needed to identify biomarkers of effects for specific target organs following exposure to fuel oils. 

Antibodies were generated against a bent A-alky 1 mesoporpbyrin, which is a known inhibitor of the enzyme ferrochelatase. Ferrochelatase catalyzes the insertion of Fe(II) into protoporphyrin, as part of the heme biosynthetic pathway (Lavallee, 1988). Antibody 7G12 was found to catalyze the insertion of divalent metal ions into porphyrins, with a rate similar to thatfound for ferrochelatase (Cochran and Schultz, 1990). The structural data presented below supports the hypothesis that the transition state for porphyrin metallation involves a distortion of the macrocyclic ring system to facilitate metal insertion, and that this bent porphyrin acts as a good transition-state mimic in the development of catalytic antibody 7G12. 

A second major lead-induced toxicity involves interruption of heme synthesis. Lead interacts at several steps in the heme biosynthetic pathway (Figure 21.13). As mentioned above, Pb inhibits the enzyme 8-aminolevulinic acid dehydratase (ALA-D), which catalyzes the second step of heme synthesis involving the condensation of two molecules of aminolevulinic acid (ALA) to form porphobilinogen. The result of this inhibition is the accumulation of aminolevulinic acid in the serum and increased excretion of ALA in the urine. A second major disruption of the heme biosynthetic pathway is Pb inhibition of ferrochelatase. This enzyme is responsible for the incorporation of the ferrous ion (Fe2+) into protoporphrin IX to produce heme (Figure 21.2). Accumulated protoporphrin is incorporated into red blood cells and chelates zinc as the cells circulate. This zinc-protoporphrin complex is fluorescent and used to diagnose Pb poisoning. 

Aminolevulinic acid dehydrase (ALA dehydrase) is the second enzyme of the heme biosynthetic pathway. It catalyzes the condensations of two molecules of ALA to form porphyrobilinogen (PBG). 

Garcia-Vargas, G.G., Hemandez-Zavala, A. (1996). Urinary porphyrins and heme biosynthetic enzyme activities measured by HPLC in arsenic toxicity. Biomed. Chromatogr. 10 278-84. 

An example of this approach is demonstrated in an antibody mimic of the enzyme ferrochetalase (39). Ferrochelatase catalyzes the insertion of Fe + into protoporphyrin IX (3) as the last step in the heme biosynthetic pathway (40). Interestingly, N-alkylporphyrins are known to be potent inhibitors of this enzyme, because alkylation at one pyrrole lutrogen distorts the planarity of the porphyrin macrocycle (41). This finding was used in the design of hapten 4 to catalyze the incorporation of metal ions into mesoporphyrin IX (5) by eliciting an antibody that binds the substrate in a ring-strained conformation. 

Hydroxymethylbilane Synthase (EC 2.5.1.61), HMBS HMBS (also known as porphobilinogen [PEG] deaminase) is a cytoplasmic enzyme that catalyzes the formation of one molecule of the linear tetrapyrrole 1-hydroxymethylbilane (HMB also known as preuroporphyrinogen) from four molecules of PEG with the release of four molecules of ammonia. The former enzyme committee designation for HMBS was EC 4.3.1.8, but in 2003 the enzyme was redesignated as EC 2.5.1.61. The enzyme has two molecules of its own substrate PEG, attached covalently to the apoenzyme as a prosthetic group. The enzyme is susceptible to allosteric inhibition by intermediates further down the heme biosynthetic pathway, notably coproporphyrinogen-III and protoporphyrinogen-IX. 

Assay of the individual enzymes of the heme biosynthetic pathway is rarely required for the investigation of patients with symptoms of porphyria. However, measurement of enzyme activities is useful for family studies when the individual mutation cannot be identified or when DNA analysis is not available, and for the identification of subtypes such... 

Heme biosynthetic pathway and the enzyme defects in various porphyrias. AD, autosomal dominant AR, autosomal recessive. 

Porphyrias are inherited or acquired disorders caused by a deficiency of enzymes in the heme biosynthetic pathway. Porphyrin is synthesized in both the erythroblasts and the liver, and either one may be the site of a disorder. Congenital erythropoietic pOTjdtyria, for example, prematurely destroys eythrocytes. This disease results from insufficient cosynthase. In this porphyria, the synthesis of the required amount of uroporphyrinogen III is accompanied by the formation of very large quantities of uroporphyrinogen I, the useless symmetric isomer. Uroporphyrin I, coproporphyrin I, and other symmetric derivatives also accumulate. The urine of... 

Figure 44-3. Heme biosynthetic pathway and characteristics associated with specific enzyme-deficiency porphyrias. ADP = ALA dehydratase deficiency porphyria AIP = acute intermittent porphyria CEP = congenital erythropoietic porphyria PCT = porphyria cutanea tarda HEP = hepatoerythropoietic porphyria HCP = hereditary coproporphyria VP = variegate porphyria EPP = erythropoietic protoporphyria.

The first step of the heme biosynthetic pathway in mammalian cells involves the condensation of glycine with succinyl-coenzyme A (CoA) to yield 5-aminolevulinate (ALA), carbon dioxide and CoA (Figure 2-1). This reaction is catalyzed by ALA synthase (ALAS E.C. 2.3.1.37) and is considered to be the rate-limiting step in the production of heme in, at least, non-erythroid cells [1, 7, 8]. ALAS was initially discovered in the bacterimn Rhodobacter spheroides and in cMcken erythrocytes in the laboratories of Shemin [9] and Neuberger [10], respectively. However, it was not imtil the 1970s that the enzyme started to be isolated and purified from mammalian sources [11-13]. 

The rate of synthesis of erythroid ALAS, the first enzyme of the heme biosynthetic pathway, is directly dependent on the cellular iron concentration. Clinically, mutations in the ALAS2 gene are associated with XLSA. Although the cellular iron uptake systems remain completely functional in XLSA patients, this type of side-... 

Heme, the most abundant iron cofactor, can play diversified roles in the cell. These roles include not only the already-mentioned regulatory and signal transduction processes, but also electron transfer, oxygen binding and transport, and direct involvement in the oxygen metabolism. The first step of the heme biosynthetic pathway in mammalian cells is catalyzed by 5-aminolevulinic acid synthase (ALAS), which is considered a rate-limiting step in the production of heme. The rate of synthesis of erythroid ALAS is directly dependent on the cellular iron concentration. Ferreira reviews recent structural and site-directed mutagenesis studies on ALAS (Chapter 2), which, for example, have revealed that the homodimeric enzyme s active site is located at the subunit interface and contains catalytically essential residues from both subunits. 

A [2Fe-2S] cluster is also present in mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, and it appears to be the determinant for catalysis as reviewed by Franco et al. (Chapter 3). Nevertheless, the specific role of this cluster in the enzyme s stability and function remains to be established. The search for the function of this cluster becomes even more challenging as bacterial, yeast and plant ferrochelatases do not possess it, although they perform the same catalytic task. 

Each type of porphyria (indicated in Figure 2 by a name on a white background) is associated with abnormal levels of activity of a specific enzyme in the heme biosynthetic pathway [67-91]. For example, a reduction in ferrochelatase activity is associated with protoporphyria, a condition in which photosensitizing concentrations of PpIX may accumulate because it is being produced faster than it can be converted into heme [92]. 

The major mechanism responsible for tissue-to-tissue differences in the accumulation of ALA-induced PpIX almost certainly involves differences in the enzyme profile of the heme biosynthetic pathway. For example, a large enough alteration in the maximum biosynthetic capacity of any of the steps upstream from PpIX would alter the capacity of the pathway as a whole to synthesize PpIX, while a significant alteration downstream from PpIX would lead to an alteration in the rate of conversion of PpIX into heme. 

Figure 30. One of the best biomarkers for lead poisoning is the second enzyme in the heme biosynthetic pathway, ALAD, which is inhibited by femtomolar concentrations of lead (467). Activity of ALAD in red blood cells correlates inversely with blood lead level. [Figure reprinted with permission from Elsevier Science (J. A. Millar, V. Battistini, R. L. Gumming, F. Carswell, and A. Goldberg, The Lancet, 1970, Vol. 2, 695-698).]...

Several other classes of proteins have also been implicated as possible targets for lead, including other proteins in the heme biosynthetic pathway, leadbinding proteins in the kidney and brain, and heat shock proteins (342, 500-502). Lead is known to affect several steps in the heme biosynthetic pathway other than that catalyzed by ALAD Other profound effects include stimulation of 5-aminolevulinic acid synthase (ALAS) and decreased levels of iron incorporation into protoporphyrin by ferrochelatase (see Section VI.E.2 and Fig. 34) (10, 503-505). However, not all of these effects are due to direct interactions between lead and enzymes in the heme biosynthetic pathway. For instance, the widespread assertion that lead inhibits ferrochelatase is not supported by studies on the isolated enzyme (506, 507). Furthermore, increased levels of both erythrocyte protoporphyrin IX (EP) and zinc protoporphyrin (ZPP) are observed at high BLLs, suggesting that ferrochelatase is stiU competent to insert zinc into EP and that the increased levels of EP and ZPP associated with lead poisoning are most likely caused by lead interfering with iron uptake or transport (see Sections VI.C.4 and VI.E) (10, 506, 507). 

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