Uroporphyrinogen decarboxylase. Purification, properties, and inhibition by polychlorinated biphenyl isomers

Uroporphyrinogen decarboxylase (EC 4.1.1.37) which converts uroporphyrinogen I or III into coproporphyrinogen I or III, respectively, was purified about 5,500-fold from chicken erythrocytes. Purification was accomplished by chromatography on DEAE-cellulose, ammonium sulfate fractionation, chromatography on Sephadex G-100, and chromatofocusing. The most purified preparation was homogeneous on polyacrylamide gel electrophoresis and had a specific activity of 1,420 units/mg of protein, the highest value so far reported. The molecular weight, as determined by Sephadex G-150 gel chromatography, is 79,000. The subunit molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 39,700, suggesting that uroporphyrinogen decarboxylase is dimeric in form. The purified enzyme had an isoelectric point of 6.2 and a pH optimum of 6.8. The SH reagents inhibited the enzyme activity, but neither metal ions nor cofactor requirements could be demonstrated. A new and simple method for the separation of free uroporphyrin, hepta-, hexa-, and pentacarboxylic porphyrins and coproporphyrin was developed using a high pressure liquid chromatograph equipped with a spectrofluorometric detector. Kinetic studies of the sequential decarboxylation of uroporphyrinogen with purified enzyme were performed. 3,4,3',4'-Tetrachlorobiphenyl and 3,4,5,3',4'5'-hexachlorobiphenyl which specifically induce delta-aminolevulinic acid synthetase also strongly inhibit uroporphyrinogen decarboxylase directly at two steps, i.e. first in the formation of hexacarboxylic porphyrinogen III from heptacarboxylic porphyrinogen III and second in the formation of heptacarboxylic porphyrinogen III from uroporphyrinogen III.     

Investigations of rat liver uroporphyrinogen decarboxylase. Comparisons of porphyrinogens I and III as substrates and the inhibition by porphyrins.

1. The decarboxylations of uroporphyrinogens, hepta-, hexa- and penta-carboxyporphyrinogens I and III by porphyrinogen carboxy-lyase (EC 4.1.1.37) in rat liver supernatant have been compared as functions of substrate concentrations. Although Km and Vmax. (for total porphyrinogens formed) were estimated, prophyrinogens and CO2 produced at 1 microM were considered to be a better indication of real relative rates, owing to substrate/product inhibitions. Uroporphyrinogen III was the best substrate by the criteria of Km/Vmax. and decarboxylation at 1 microM and was converted into coproporphyrinogen more quickly than its series-I isomer. 2. The difference between uroporphyrinogens I and III as substrates was confirmed by using a mixture of [14C8]uroporphyrinogens, the discrimination occurring principally in the first decarboxylation. 3. Porphyrins, especially oxidation products of the substrates, inhibited the enzyme. Heptacarboxyporphyrin III was the most effective inhibitor of both uroporphyrinogen III and heptacarboxyporphyrinogen III conversion into coproporphyrinogen. 4. Rapid analysis of the livers from rats made porphyric with hexachlorobenzene demonstrated that substantial quantities of the tetrapyrroles were present in vivo as the porphyrinogens (21-42%). 5. Enzymic decarboxylation of uroporphyrinogen III in 2H2O-containing buffer gave [2H4]coproporphyrinogen. 6. Rats treated with cycloheximide for 10h showed no decrease in uroporphyrinogen decarboxylase activity/mg of protein, suggesting a relatively slow turnover of the enzyme.     

In vitro studies of the mechanism of inhibition of rat liver uroporphyrinogen decarboxylase activity by ferrous iron under anaerobic conditions

Human porphyria cutanea tarda (PCT) is an unusual consequence of common hepatic disorders such as alcoholic liver disease and iron overload, where hepatic iron plays a key role in the expression of the metabolic lesion, i.e., defective hepatic decarboxylation of porphyrinogens. In this investigation, kinetic studies on a partially purified rat liver uroporphyrinogen decarboxylase have been conducted under controlled conditions to determine how iron perturbs porphyrinogen decarboxylation in vitro. The enzyme, assayed strictly under anaerobic conditions in the dark, was inhibited progressively by ferrous iron. Approximately 0.45 mM ferrous ammonium sulfate was required to observe about 50% inhibition of enzyme activity measured with uroporphyrinogen I as substrate. We showed that (a) all the steps of enzymatic decarboxylation (octa-, hepta-, hexa-, and pentacarboxylic porphyrinogen of isomer I series) were inhibited by ferrous iron. The inhibition was competitive with respect to uroporphyrinogen I and III substrates; (b) the cations, e.g., Fe3+ and Mg2+, had no effect, whereas sulfhydryl group specific cations and compounds such as Hg2+, Zn2+, p-mercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoate) all inhibited the enzyme; (c) the enzyme could be protected from inhibition by Fe2+ and p-mercuribenzoate by preincubation with pentacarboxylic porphyrinogen, a natural substrate and competitive inhibitor. These data suggest for the first time a direct interaction of ferrous iron with cysteinyl residue(s) located at the active site(s) of the enzyme.     

Comparison of two assay methods for activities of uroporphyrinogen decarboxylase and coproporphyrinogen oxidase

Uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen oxidase (copro'gen oxidase) are two of the least well understood enzymes in the heme biosynthetic pathway. In the fifth step of the pathway, UROD converts uroporphyrinogen III to coproporphyrinogen III by the decarboxylation of the four acetic acid side chains. Copro'gen oxidase then converts coproporphyrinogen III to protoporphyrinogen IX via two sequential oxidative decarboxylations. Studies of these two enzymes are important to increase our understanding of their mechanisms. Assay comparisons of UROD and copro'gen oxidase from chicken blood hemolysates (CBH), using a newly developed micro-assay, showed that the specific activity of both enzymes is increased in the micro-assay relative to the large-scale assay. The micro-assay has distinct advantages in terms of cost, labor intensity, amount of enzyme required, and sensitivity.     

Studies on uroporphyrinogen decarboxylase of etiolated Euglena gracilis Z

1. A 423-fold purified fraction of uroporphyrinogen decarboxylase (EC 4.1.1.37) showing a specific activity of 770 units/mg protein has been employed in order to study some properties in etiolated Euglena gracilis Z. 2. Uroporphyrinogen decarboxylase has a relative molecular mass of 54,000, an optimum pH of 7.2 and exhibits Michaelis-Menten kinetics, employing both uroporphyrinogen I and uroporphyrinogen III as substrates. 3. Anaerobic conditions seem not to be necessary for uroporphyrinogen decarboxylase activity. Neither EDTA nor cysteine affected enzyme activity, whereas dithiothreitol produced a remarkable activation of coproporphyrinogen formation. 4. Kinetic data employing both substrates showed an accumulation of porphyrinogen (i.e. hexa- and hepta-porphyrin) containing six or seven COOH groups, depending on the uroporphyrinogen concentration used. 5. An unusual elution profile of the intermediates on Sephacryl S-200 was found.     

Uroporphyrinogen decarboxylase

Uroporphyrinogen decarboxylase (EC 4.1.1.37) catalyzes the decarboxylation of uroporphyrinogen III to coproporphyrinogen III. The amino acid sequences, kinetic properties, and physicochemical characteristics of enzymes from different sources (mammals, yeast, bacteria) are similar, but little is known about the structure/function relationships of uroporphyrinogen decarboxylases. Halogenated and other aromatic hydrocarbons cause hepatic uroporphyria by decreasing hepatic uroporphyrinogen decarboxylase activity. Two related human porphyrias, porphyria cutanea tarda and hepatoerythropoietic porphyria, also result from deficiency of this enzyme. The roles of inherited and acquired factors, including iron, in the pathogenesis of human and experimental uroporphyrias are reviewed.     

Purification of uroporphyrinogen decarboxylase from human erythrocytes. Immunochemical evidence for a single protein with decarboxylase activity in human erythrocytes and liver.

Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified 4419-fold to a specific activity of 58.3 nmol of coproporphyrinogen III formed/min per mg of protein (with pentacarboxyporphyrinogen III as substrate) from human erythrocytes by adsorption to DEAE-cellulose, (NH4)2SO4 fractionation, gel filtration, phenyl-Sepharose chromatography and polyacrylamide-gel electrophoresis. Progressive loss of activity towards uroporphyrinogens I and III occurred during purification. Experiments employing immunoprecipitation, immunoelectrophoresis and titration with solid-phase antibody indicated that all the uroporphyrinogen decarboxylase activity of human erythrocytes resides in one protein, and that the substrate specificity of this protein had changed during purification. The purified enzyme had a minimum mol.wt. of 39 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gel filtration gave a mol.wt. of 58 000 for the native enzyme. Isoelectric focusing showed a single band with a pI of 4.60. Reaction with N-ethylmaleimide abolished both catalytic activity and immunoreactivity. Incubation with substrates or porphyrins prevented inactivation by N-ethylmaleimide. An antiserum raised against purified erythrocyte enzyme precipitated more than 90% of the uroporphyrinogen decarboxylase activity from human liver. Quantitative immunoprecipitation and crossed immunoelectrophoresis showed that the erythrocyte and liver enzymes are very similar but not identical. The differences observed may reflect secondary modification of enzyme structure by proteolysis or oxidation of thiol groups, rather than a difference in primary structure.     

Purification and characterization of uroporphyrinogen decarboxylase from tobacco leaves

1. Uroporphyrinogen decarboxylase which catalyzes the formationof coproporphyrinogen from uroporphyrinogen is located in thesoluble fraction of tobacco leaves and was purified 72 foldthrough ammonium sulphate precipitation and calcium phosphosphategel absorption. 2. Kinetic studies indicated that the apparentMichaelis constant was 1 ? 10^-6 M for uroporphyrinogen III (pH6.5; 37?C). Uroporphyrinogen III served as a much better substratethan uroporphyrinogen I under the standard conditions of thisstudy. 3. Enzyme activity was inhibited by thiol reagents andheavy divalent cations and was stimulated by some chelatingagents. 4. Both chloride and fluoride salts inhibited the formationof coproporphyrinogen from uroporphyrinogen. ¹Present address: Department of Chemistry, Simon Fraser University,Burnaby 2, British Columbia, Canada. ²Present address: Biology Department, Utah State University,Logan, Utah 84322, U. S. A. (Received June 8, 1974; )     

Evidence for two uroporphyrinogen decarboxylase isoenzymes in human erythrocytes

In animals and plants, uroporphyrinogen decarboxylase catalyzes the stepwise decarboxylations of uroporphyrinogen, the precursor of heme and chlorophyll. To better understand its metabolic roles, we characterized the enzyme purified to electrophoretic homogeneity (about 11,000-fold) from human erythrocytes by a novel uroporphyrin-sepharose affinity chromatographic method. Native polyacrylamide disc gel electrophoresis of the purified enzyme preparation showed two bands detected by staining either for protein or with uroporphyrin-I. Each individual protein eluted from the gel when subjected to re-electrophoresis on SDS-polyacrylamide gel, appeared as a single protein band with molecular masses of approximately 54,000 and approximately 35,000 daltons respectively. Both proteins were able to catalyze all four decarboxylation steps, though the ratios of enzyme activity using octa-, hepta-, hexa- to pentacarboxylic porphyrinogen substrates were distinctly different. Also, their kinetic analysis with heptacarboxylic porphyrinogen-I substrate provided distinctly different apparent Michaelis constants. This provides the first evidence that decarboxylations of uroporphyrinogen to coproporphyrinogen are catalyzed by two isoenzymes.     

Purification and properties of uroporphyrinogen III synthase from human erythrocytes

Uroporphyrinogen III synthase (hydroxymethylbilane hydro-lyase (cyclizing); EC 4.2.1.75), the fourth enzyme in the heme biosynthetic pathway, was purified to homogeneity from human erythrocytes. For enzyme purification and characterization, a sensitive coupled enzyme assay was used which generated the substrate, hydroxymethylbilane; the oxidized product, uroporphyrin III, was quantitated by high pressure liquid chromatography. Uroporphyrinogen III synthase was initially separated from delta-aminolevulinate dehydratase and hydroxymethylbilane synthase by a preparative anion exchange chromatographic step. Subsequent chromatography on hydroxyapatite, phenyl-Sepharose, and Sephadex G-100 purified the enzyme about 70,000-fold with an 8% yield. Homogeneous enzyme was obtained following a final C4-reversed phase high pressure liquid chromatographic step which removed a single major and several minor protein contaminants from the enzyme. The purified enzyme had a specific activity of over 300,000 units/mg, an isoelectric point of 5.5, and was thermolabile (t1/2 at 60 degrees C approximately 1 min). Molecular weight studies by gel filtration (Mr approximately equal to 30,000) and analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately equal to 29,500) were consistent with the enzyme being a monomer. Using hydroxymethylbilane as substrate, the purified enzyme formed uroporphyrinogen III in the absence of hydroxymethylbilane synthase or other cofactors. The pH optimum was 7.4 and the Km for hydroxymethylbilane was 5-20 microM. The enzyme was activated by Na+, K+, Mg+, and Ca2+ and was inhibited by Cd2+, Cu2+, Hg2+, and Zn2+. Amino acid composition analysis was performed, and the N-terminal sequence, Met-Lys-Val-Leu-Leu-Leu, was determined by microsequencing. The availability of the purified enzyme should permit investigation of its reaction mechanism as well as facilitate biochemical and molecular studies of the genetic defect in congenital erythropoietic porphyria.     

Oxygen-dependent coproporphyrinogen III oxidase (HemF) from Escherichia coli is stimulated by manganese

During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Oxygen-dependent coproporphyrinogen III oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. The dimeric enzyme showed a Km value of 2.6 microm for coproporphyrinogen III with a kcat value of 0.17 min-1 at its optimal pH of 6. HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. Molecular oxygen is essential for the enzymatic reaction. Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H2O2. Metal chelator treatment inactivated E. coli HemF. Only the addition of manganese fully restored coproporphyrinogen III oxidase activity. Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained. One catalytically important tryptophan residue was localized in position 274. None of the tested highly conserved cysteine (Cys-167), tyrosine (Tyr-135, Tyr-160, Tyr-170, Tyr-213, Tyr-240, and Tyr-276), and tryptophan residues (Trp-36, Trp-123, Trp-166, and Trp-298) were found important for HemF activity. Moreover, mutation of a potential nucleotide binding motif (GGGXXTP) did not affect HemF activity. Two alternative routes for HemF-mediated catalysis, one metal-dependent, the other metal-independent, are proposed.     

Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans.

S-Adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT), the enzyme of the cobalamin biosynthetic pathway which catalyzes C methylation of uroporphyrinogen III, was purified about 150-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans derived from a cobalamin-overproducing strain by ammonium sulfate fractionation, anion-exchange chromatography, and hydroxyapatite chromatography. The purified protein has an isoelectric point of 6.4 and molecular weights of 56,500 as estimated by gel filtration and 30,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer. It does not contain a chromophoric prosthetic group and does not seem to require metal ions or cofactors for activity. SUMT catalyzes the two successive C-2 and C-7 methylation reactions involved in the conversion of uroporphyrinogen III to precorrin-2 via the intermediate formation of precorrin-1. In vitro studies suggest that the intermediate monomethylated product (precorrin-1) is released from the protein and then added back to the enzyme for the second C-methylation reaction. The pH optimum was 7.7, the Km values for S-adenosyl-L-methionine and uroporphyrinogen III were 6.3 and 1.0 microM, respectively, and the turnover number was 38 h-1. The enzyme activity was shown to be completely insensitive to feedback inhibition by cobalamin and corrinoid intermediates tested at physiological concentration. At uroporphyrinogen III concentrations above 2 microM, SUMT exhibited a substrate inhibition phenomenon. It is suggested that this property might play a regulatory role in cobalamin biosynthesis in the cobalamin-overproducing strain studied.     

The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase activity in mitochondrial extracts of Saccharomyces cerevisiae.

The oxidation of protoporphyrinogen IX to protoporphyrin IX in yeast cells is enzyme-dependent. The enzyme, protoporphyrinogen oxidase, associated with purified mitochondria isolated from Saccharomyces cerevisiae was solubilized by sonic treatment in the presence of detergent and partially purified. The molecular weight of the enzyme was 180,000 plus or minus 18,000. The purified preparation could be stored at -20 degrees in the presence of 20% glycerol for several months without loss of activity. Enzyme activity was destroyed by heating above 40 degrees and by proteolytic digestion and irreversible inactivation occurred outside the pH range of 4.0 to 9.5. The pH optimum of the enzymic reaction was 7.45 and the value of the Michaelis constant was approximately 4.8 muM. Protoporphyrinogen oxidase did not catalyse the oxidation of coproporphyrinogen I or III or uroporphyrinogen I or III and the rate of enzymic oxidation of mesoporphyrinogen IX was less than 20% of that observed with protoporphyrinogen IX. The presence of thiol groups in the enzyme system was indicated but no metal ion or other cofactor requirement was demonstrated. Enzyme activity was insensitive to cyanide, 2,4-dinitrophenol, and azide whereas it was inhibited in the presence of Cu-2+ or Co-2+ ions, high ionic strength, heme, or hemin.     

Porphyria-induced hepatic porphyrinogen carboxy-lyase inhibitor and its interaction with the active site(s) of the enzyme.

Porphyrinogen carboxy-lyase is an enzyme that sequentially decarboxylates uroporphyrinogen III (8-COOH) to yield coproporphyrinogen III (4-COOH). In mammals this enzyme activity is impaired by hexachlorobenzene treatment, through generation of an enzyme inhibitor. The interaction of porphyrinogen carboxy-lyase inhibitor, extracted from the liver of hexachlorobenzene-treated rats, with substrate decarboxylation sites on the enzyme, was studied using four different carboxylated substrates belonging to the isomeric III series of naturally-formed porphyrinogens containing 8-,7-,6- and 5-COOH. Similar inhibitor effects were elicited against all the substrates assayed, with the exception of pentacarboxyporphyrinogen III in which decarboxylation was not inhibited to same extent. Enzyme protection assays in the presence of the different substrates, indicated that each porphyrinogen protects its own decarboxylation from inhibitor action. Preincubation of the inhibitor with normal enzyme increased its inhibitory effect. On the other hand, preincubation of both enzyme and inhibitor with superoxide dismutase or mannitol, did not alter inhibitory activity. Preincubation of the inhibitor with a number of amino acids showed that only arginine and its derivative N alpha-Benzoyl-L-Arginine ethyl ester interact with the inhibitor, noticeably reducing its ability to inhibit porphyrinogen carboxy-lyase. Albumin, histidine, serine, cysteine and imidazol, were unable to quench inhibitor activity. The present results indicate that the inhibitor acts at the binding site of each porphyrinogen. Taking into account that arginine is related to enzyme activity, and that histidine is found at the binding site of the substrates, the results suggest that the inhibitor could bind to arginine residues, blocking the access of substrates to histidine and altering the adequate orientation for decarboxylation by masking the positively charged active site necessary for porphyrinogen binding to the enzyme. In addition an indirect effect of the inhibitor mediated through free radicals could be discarded.     

Genetic and biochemical characterization of mutants of Saccharomyces cerevisiae blocked in six different steps of heme biosynthesis

Summary Heme-deficient mutants of Saccharomyces cerevisiae have been isolated from two isogenic strains with the use of an enrichment method based on photodynamic properties of Zn-protoporphyrin. They defined seven non-overlapping complementation groups. A mutant representative of each group was further analysed. Genetic analysis showed that each mutant carried a single nuclear recessive mutation. Biochemical studies showed that the observed accumulation and/or excretion of the different heme synthesis precursors by the mutant cells correlated well with the enzymatic deficiencies measured in acellular extracts. Six of the seven mutants were blocked in a different enzyme activity: 5-aminolevulinate synthase, porphobilinogen synthase, uroporphyrinogen I synthase, uroporphyrinogen decarboxylase, coproporphyrinogen III oxidase and ferrochelatase. The other mutant had the same phenotype as the mutant deficient in ferrochelatase activity. However, it possessed a normal ferrochelatase activity when measured in vitro, so this mutant was assumed to be deficient in protoporphyrinogen oxidase activity or in the transport and/or reduction of iron.The absence of PBG synthesis led to a total lack of uroporphyrinogen I synthase activity. The absence of heme, the end product, led to an important increase of coproporphyrinogen III oxidase activity, while the activity of 5-aminolevulinate synthase, the first enzyme of the pathway, was not changed. These results are discussed in terms of possible modes of regulation of heme synthesis pathway in yeast.     

Unique properties of Plasmodium falciparum porphobilinogen deaminase

The hybrid pathway for heme biosynthesis in the malarial parasite proposes the involvement of parasite genome-coded enzymes of the pathway localized in different compartments such as apicoplast, mitochondria, and cytosol. However, knowledge on the functionality and localization of many of these enzymes is not available. In this study, we demonstrate that porphobilinogen deaminase encoded by the Plasmodium falciparum genome (PfPBGD) has several unique biochemical properties. Studies carried out with PfPBGD partially purified from parasite membrane fraction, as well as recombinant PfPBGD lacking N-terminal 64 amino acids expressed and purified from Escherichia coli cells (DeltaPfPBGD), indicate that both the proteins are catalytically active. Surprisingly, PfPBGD catalyzes the conversion of porphobilinogen to uroporphyrinogen III (UROGEN III), indicating that it also possesses uroporphyrinogen III synthase (UROS) activity, catalyzing the next step. This obviates the necessity to have a separate gene for UROS that has not been so far annotated in the parasite genome. Interestingly, DeltaPfP-BGD gives rise to UROGEN III even after heat treatment, although UROS from other sources is known to be heat-sensitive. Based on the analysis of active site residues, a DeltaPfPBGDL116K mutant enzyme was created and the specific activity of this recombinant mutant enzyme is 5-fold higher than DeltaPfPBGD. More interestingly, DeltaPfPBGDL116K catalyzes the formation of uroporphyrinogen I (UROGEN I) in addition to UROGEN III, indicating that with increased PBGD activity the UROS activity of PBGD may perhaps become rate-limiting, thus leading to non-enzymatic cyclization of preuroporphyrinogen to UROGEN I. PfPBGD is localized to the apicoplast and is catalytically very inefficient compared with the host red cell enzyme.     

Substrate Shuttling between Active Sites of Uroporphyrinogen Decarboxylase Is Not Required to Generate Coproporphyrinogen

Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.     

Liver porphyrinogen carboxylase in hexachlorobenzene porphyric rats. Studies with intermediate porphyrinogens of series III and with uroporphyrinogen I

The present work studies the action of hexachlorobenzene (HCB) on the decarboxylation of uroporphyrinogen (Urogen) I and III and also on the decarboxylation of intermediate porphyrinogens of series III under different conditions using liver of normal and porphyric rats as enzyme source. The same enzyme is involved in the Urogen decarboxylation of both isomeric series I and III and catalyses the four steps in both cases. HCB affects all of them. HCB blocks the four steps of Urogen III decarboxylation to the same degree, as a function of intoxication time. HCB leads, in general, to an increase in the efficiency (Km/Vmax) of the porphyric system. These data can be interpreted as a reaction of the organism to overcome the enzymatic blockade.     

Abnormal kinetic behavior of uroporphyrinogen decarboxylase obtained from rats with hexachlorobenzene-induced porphyria

Uroporphyrinogen decarboxylase is an essential enzyme in all organisms and functions in the heme biosynthetic pathway, catalyzing the decarboxylation of the four acetate groups of uroporphyrinogen to form coproporphyrinogen. This work examines whether the four sequential decarboxylations occur at the same active site, and explores whether hexachlorobenzene-induced porphyria affects the behavior of the enzyme. For this purpose, kinetic competition studies were done with mixtures of uroporphyrinogen III and pentacarboxyporphyrinogen III. With the enzyme from normal rats, a constant velocity was obtained with all the mixtures, indicating that uroporphyrinogen and pentacarboxy-porphyrinogen react at the same active site, i.e. the first and fourth decarboxylations occur at the same site. In contrast, in experiments with enzyme from rats with hexachlorobenzene-induced porphyria, the total rate for mixtures was always lower than the reference rate; and a curve with a deep minimum was obtained, indicating that the two reactions occur at functionally different sites, but with cross-inhibition. This suggests that the modifications induced in the enzyme by hexachlorobenzene cause the two active sites to become nonequivalent and functionally different. The question is discussed how the hexachlorobenzene treatment may produce this abnormal kinetic behavior, and alternative hypotheses are considered.