Random decarboxylation of uroporphyrinogen III by human hepatic uroporphyrinogen decarboxylase

The type III heptacarboxylic porphyrinogens derived from enzymic decarboxylation of an acetic acid substituent on uroporphyrinogen III to a methyl group by human hepatic uroporphyrinogen decarboxylase has been analysed by reversed-phase high-performance liquid chromatography with electrochemical detection. The results showed that all four possible heptacarboxylic acid porphyrinogen isomers, with the methyl group attached to rings A, B, C and D of the tetrapyrrole macrocycle, respectively, were formed in almost equal proportions. It was concluded that the normal pathway of uroporphyrinogen III decarboxylation in human liver follows a random mechanism.     

Decarboxylation of porphyrinogens by rat liver uroporphyrinogen decarboxylase.

The decarboxylations of uroporphyrinogens I and III and of heptacarboxylic, hexacarboxylic and pentacarboxylic porphyrinogens III by rat liver uroporphyrinogen decarboxylase were compared, and the results suggest that the removal of the first carboxy group from uroporphyrinogen III is a more rapid step than that from isomer I or the other substrates investigated.     

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.     

Oxidation of uroporphyrinogens by hydroxyl radicals. Evidence for nonporphyrin products as potential inhibitors of uroporphyrinogen decarboxylase

A hydroxyl radical-generating system, hypoxanthine/xanthine oxidase/Fe-EDTA, oxidises uroporphyrinogens to uroporphyrins and to more polar nonporphyrin products. The evidence suggests that the nonporphyrin products have inhibitory activity towards mouse liver uroporphyrinogen decarboxylase which could explain some forms of human and experimental porphyrias.     

Structural basis for tetrapyrrole coordination by uroporphyrinogen decarboxylase

Uroporphyrinogen decarboxylase (URO-D), an essential enzyme that functions in the heme biosynthetic pathway, catalyzes decarboxylation of all four acetate groups of uroporphyrinogen to form coproporphyrinogen. Here we report crystal structures of URO-D in complex with the I and III isomer coproporphyrinogen products. Crystallization required use of a novel enzymatic approach to generate the highly oxygen-sensitive porphyrinogen substrate in situ. The tetrapyrrole product adopts a domed conformation that lies against a collar of conserved hydrophobic residues and allows formation of hydrogen bonding interactions between a carboxylate oxygen atom of the invariant Asp86 residue and the pyrrole NH groups. Structural and biochemical analyses of URO-D proteins mutated at Asp86 support the conclusion that this residue makes important contributions to binding and likely promotes catalysis by stabilizing a positive charge on a reaction intermediate. The central coordination geometry of Asp86 allows the initial substrates and the various partially decarboxylated intermediates to be bound with equivalent activating interactions, and thereby explains how all four of the substrate acetate groups can be decarboxylated at the same catalytic center.     

Decarboxylation of uroporphyrinogen I and III in 2,3,7,8-tetrachlorodibenzo-p-dioxin induced porphyria in mice

• 1.1. The decarboxylation of uroporphyrinogens I and III by porphyrinogen carboxy-lyase (EC 4.1.1.37) in mouse liver supernatant was compared in relation to substrate concentrations.
• 2.2. In this species uroporphyrinogen III was the best substrate judging by the criteria of K_m/V_max (estimated for total porphyrinogens) and was converted into coproporphyrinogen faster than its series I isomer.
• 3.3. The difference between the two isomers was mainly due to the first decarboxylation.
• 4.4. This difference was confirmed by calculation of the Hill coefficient and of Lineweaver-Burk plot which suggested that isomer I induced negative cooperativity in the active centre of the enzyme.
• 5.5. After treatment with a porphyrogenic dose of TCDD (25 μg/kg/week for 9 weeks) differences between uroporphyrinogen I and III as substrate were maintained.
• 6.6. In addition treatment reduced V_max and K_m (estimated for total porphyrinogens) of liver porphyrinogen carboxy-lyase to about half control values for both isomers.
• 7.7. V_max was reduced mainly because of the formation of smaller amounts of all products of decarboxylation, and K_m because more heptaporphyrinogen was formed than coproporphyrinogen.
• 8.8. Values of the Hill coefficient and Lineweaver-Burk plots suggested TCDD induced altered substrate affinity for isomer III too.
• 9.9. Treatment with TCDD did not affect the decarboxylation of uroporphyrinogen III by RBC porphyrinogen carboxy-lyase, estimated from K_m and V_max for total porphyrinogens formed.

     

Crystal structure of human uroporphyrinogen decarboxylase.

Uroporphyrinogen decarboxylase (URO-D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. This activity is essential in all organisms, and subnormal activity of URO-D leads to the most common form of porphyria in humans, porphyria cutanea tarda (PCT). We have determined the crystal structure of recombinant human URO-D at 1.60 A resolution. The 40.8 kDa protein is comprised of a single domain containing a (beta/alpha)8-barrel with a deep active site cleft formed by loops at the C-terminal ends of the barrel strands. Many conserved residues cluster at this cleft, including the invariant side chains of Arg37, Arg41 and His339, which probably function in substrate binding, and Asp86, Tyr164 and Ser219, which may function in either binding or catalysis. URO-D is a dimer in solution (Kd = 0.1 microM), and this dimer also appears to be formed in the crystal. Assembly of the dimer juxtaposes the active site clefts of the monomers, suggesting a functionally important interaction between the catalytic centers.     

Density functional models of the mechanism for decarboxylation in orotidine decarboxylase

The mechanism of orotidine 5-monophosphate decarboxylase (ODCase) has been modeled using hybrid Density Functional Theory (B3LYP functional). The main goal of the present study was to investigate if much larger quantum chemical models of the active site than previously used could shed new light on the mechanism. The models used include the five conserved amino acids expected to be the most important ones for catalysis. One result of this model is that a mechanism involving a direct cleavage of the C–C bond followed by a protonation of C6 by Lys93 appears unlikely, with a barrier for decarboxylation 20 kcal mol^–1 too high. Additional effects like electrostatic stress and ground-state destabilization have been estimated to have only a minor influence on the reaction barrier. The conclusion from the calculations is that the negative charge developing on the substrate during decarboxylation must be stabilized by a protonation of the carbonyl O2 of the substrate. For this mechanism, the addition of the catalytic amino acids decreases the reaction barrier by 25 kcal mol^–1, but full agreement with experimental results has still not been reached. Further modifications of this mechanism are discussed. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located a http://dx.doi.org/10.1007/s00894-002-0080-2.Electronic Supplementary Material available.     

Characterization and crystallization of human uroporphyrinogen decarboxylase.

The cytosolic enzyme uroporphyrinogen decarboxylase (URO-D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. Recombinant human URO-D has been expressed in Escherichia coli with a histidine tag and has been purified to homogeneity. Purified protein was determined to be a monodisperse dimer by dynamic light scattering. Equilibrium sedimentation analysis confirmed that the protein is dimeric, with a dissociation constant of 0.1 microM. URO-D containing an amino-terminal histidine tag was crystallized in space group P3(1)21 or its enantiomer P3(2)21 with unit cell dimensions a = b = 103.6 A, c = 75.2 A. There is one molecule in the asymmetric unit, consistent with generation of the dimer by the twofold axis of this crystallographic operator. Native data have been collected to 3.0 a resolution.     

Rat hepatic uroporphyrinogen III co-synthase. Purification and evidence for a bound folate coenzyme participating in the biosynthesis of uroporphyrinogen III.

Rat hepatic uroporphyrinogen III co-synthase was isolated and purified 73-fold with a 13% yield by (NH4)2SO4 fractionation and sequential chromatography on DEAE-Sephacel, Sephadex G-100 (superfine grade) and folate-AH-Sepharose 4B. The purified co-synthase has an Mr of approx. 42 000, and is resolved into two bands, each possessing co-synthase activity, by polyacrylamide-gel electrophoresis. A factor was dissociated from the purified co-synthase. Results of both microbiological and competitive protein-binding assays suggest that it is a pteroylpolyglutamate. The isolated pteroylpolyglutamate factor was co-eluted with authentic N5-methyltetrahydropteroylheptaglutamate on DEAE-Sephacel. Uroporphyrinogen III is formed by cosynthase-free preparations of uroporphyrinogen I synthase in the presence of tetrahydropteroylglutamate. Tetrahydropeteroylheptaglutamate is also able to direct the formation of equivalent amounts of uroporphyrinogen III at a concentration approximately one-hundredth that of tetrahydropteroylmonoglutamate. These results suggest that a reduced pteroylpolyglutamate factor is associated with rat hepatic uroporphyrinogen III co-synthase, and that this may function as a coenzyme for the biosynthesis of uroporphyrinogen III.     

Inhibition of uroporphyrinogen decarboxylase activity. The role of cytochrome P-450-mediated uroporphyrinogen oxidation.

It was previously shown that uroporphyrinogen oxidation is catalysed by a form of cytochrome P-450 induced by 3-methylcholanthrene [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We have now measured uroporphyrinogen oxidation and uroporphyrinogen decarboxylation simultaneously in 10,000 g supernatants from the livers of methylcholanthrene-treated mice and chick embryos incubated with an NADPH-generating system. We found that uroporphyrinogen oxidation is associated with inhibition of uroporphyrinogen decarboxylase activity. The decreased uroporphyrinogen decarboxylase activity was not due to depletion of substrate, since decarboxylase activity was not increased by a 2.6-fold increase in uroporphyrinogen. Uroporphyrinogen oxidation and the associated inhibition of decarboxylase activity were also observed with liver supernatant from methylcholanthrene-treated chick embryo; both actions required the addition of 3,3',4,4'-tetrachlorobiphenyl. Uroporphyrinogen oxidation catalysed by microsomes from a methylcholanthrene-treated mouse inhibited the uroporphyrinogen decarboxylase activity in the 100,000 g supernatant. Ketoconazole, an inhibitor of cytochrome P-450, prevented both uroporphyrinogen oxidation and the inhibition of uroporphyrinogen decarboxylation. The addition of ketoconazole to mouse supernatant actively oxidizing uroporphyrinogen inhibited the oxidation and restored decarboxylation. The latter finding suggested that a labile inhibitor was formed during the oxidation. These results suggest uroporphyrinogen oxidation may be important in the mechanism of chemically induced uroporphyria.     

The mechanism of C-4 demethylation during cholesterol biosynthesis. Evidence for a decarboxylation mechanism not involving a Schiff-base intermediate

The conversion of 4,4-dimethylcholest-7-enol into 4alpha-methylcholest-7-enol by rat liver microsomal preparations involves the decarboxylation of a sterol 3-oxo-4alpha-carboxylic acid. By using an (18)O-labelled substrate it was shown that this decarboxylation does not involve a Schiff-base intermediate.     

Reduced substrate affinity for human erythrocyte uroporphyrinogen decarboxylase constitutes the inherent biochemical defect in porphyria cutanea tarda

The pathogenesis of human porphyria cutanea tarda (PCT) is associated with an intrinsic abnormality of the uroporphyrinogen decarboxylase enzyme. To characterize this, we studied the kinetic properties of the red cell enzyme procured from patients with various forms of PCT and non-porphyric controls. The enzyme activity (units/mg hemoglobin) in the red cell hemolysate was close to normal in sporadic PCT but about 75% diminished in the familial PCT. The Michaelis constants (Km) of 200-fold purified red cell enzyme preparations, determined by using pentacarboxylic porphyrinogen I and uroporphyrinogen I as substrates, were more than 3.8-4.0 times higher, and the maximum velocity (Vmax) was about 70% diminished in familial PCT, whereas the Km was about 1.7-1.9 times higher and the Vmax was more or less normal for sporadic PCT. These observations suggest for the first time that the primary lesion in familial PCT is a genetically determined kinetic abnormality of uroporphyrinogen decarboxylase which appears to be different from the sporadic form of the disease.     

Photodynamic inactivation of red cell uroporphyrinogen decarboxylase by porphyrins

The effects of light and porphyrins on the activity of red cell uroporphyrinogen decarboxylase were studied. Photoinactivation of uroporphyrinogen decarboxylase was dependent on uroporphyrin concentration, irradiation time and temperature. Using 40 W/m2 of UV light intensity, 40-45% decreased activity was produced with 200 microM uroporphyrin I, at 37 degrees C and after 2 hr of illumination. It has been demonstrated that porphyrins photoinactivate uroporphyrinogen decarboxylase and a mechanism for this action in relation to skin lesions is proposed.     

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.     

Kinetic mechanism of pyruvate decarboxylase. Evidence for a specific protonation of the enzymic intermediate.

Decarboxylation of pyruvate by pyruvate decarboxylase (EC 4.1.1.1) was performed in a reaction mixture containing 50% deuterium. The isolated product, acetaldehyde, was investigated directly by 1H NMR and by mass spectrometry after conversion to the 2,4-dinitrophenyl hydrazone. The protium content of 56% at acetaldehyde C1 demonstrates a specific protonation of the corresponding intermediate by the enzyme. Proton inventory studies and enzyme modification indicate the 4' amino group of the coenzyme, thiamine pyrophosphate, in an immonium structure being a possible proton donor. A 'partially concerted' mechanism is suggested for the reaction steps following the decarboxylation.     

Fern L-methionine decarboxylase: kinetics and mechanism of decarboxylation and abortive transamination

L-Methionine decarboxylase from Dryopteris filix-mas catalyzes the decarboxylation of L-methionine and a range of straight- and branched-chain L-amino acids to give the corresponding amine products. The deuterium solvent isotope effects for the decarboxylation of (2S)-methionine are D(V/K) = 6.5 and DV = 2.3, for (2S)-valine are D(V/K) = 1.9 and DV = 2.6, and for (2S)-leucine are D(V/K) = 2.5 and DV = 1.0 at pL 5.5. At pL 6.0 and above, where the value of kcat for all of the substrates is low, the solvent isotope effects on Vmax for methionine are 1.1-1.2 whereas the effects on V/K remain unchanged, indicating that the solvent-sensitive transition state occurs before the first irreversible step, carbon dioxide desorption. The enzyme also catalyzes an abortive decarboxylation-transamination reaction in which the coenzyme is converted to pyridoxamine phosphate [Stevenson, D. E., Akhtar, M., & Gani, D. (1990a) Biochemistry (first paper of three in this issue)]. At very high concentration, the product amine can promote transamination of the coenzyme. However, the reaction occurs infrequently and does not influence the partitioning between decarboxylation and substrate-mediated abortive transamination under steady-state turnover conditions. The partition ratio, normal catalytic versus abortive events, can be determined from the amount of substrate consumed by a known amount of enzyme at infinite time, and the rate of inactivation can be determined by measuring the decrease in enzyme activity with respect to time. For methionine, the values of Km as determined from double-reciprocal plots of concentration versus inactivation rate are the same as those calculated from initial catalytic (decarboxylation) rate data, indicating that a single common intermediate partitions between product formation and slow transamination. The partition ratio is sensitive to changes in pH and is also dependent upon the structure of the substrate; methionine causes less frequent inactivation than either valine or leucine. The pH dependence of the partition ratio with methionine as substrate is very similar to that for V/K. Both curves show a sharp increase at approximately pH 6.25, indicating that a catalytic group on the enzyme simultaneously suppresses the abortive reaction and enhances physiological reaction in its unprotonated state. Experiments conducted in deuterium oxide allowed the solvent isotope effects for the partition ratio and the abortive reaction to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Assay of mouse liver uroporphyrinogen decarboxylase by reverse-phase high-performance liquid chromatography

A method for the estimation of hepatic uroporphyrinogen decarboxylase activity employing reverse-phase HPLC is described. Mouse liver homogenate in 0.25 M sucrose was pretreated with a suspension of cellulose phosphate and then centrifuged to remove hemoglobin and debris. The supernatant was used as the enzyme source. Incubations were acidified, oxidized, and centrifuged only before analysis of the porphyrins formed, using a Spherisorb ODS column and a gradient solvent system constructed from methanol/lithium citrate mixtures. Coproporphyrinogen formation by BALB/c mouse liver supernatant was estimated as about 5.0 and 9.1 pmol/min/mg protein from uroporphyrinogens I and III, respectively, at 10 microM substrate concentration and pH 6.8. Decarboxylation of pentacarboxyporphyrinogens (the last step in coproporphyrinogen formation) proved to be easily measured. Coproporphyrinogen formation from pentacarboxyporphyrinogen III abd (20 microM) at pH 6.8 was about 109 pmol/min/mg protein. Pentacarboxyporphyrinogen I was not as good a substrate as III abd but was decarboxylated faster at pH 5.4 than at 6.8, and at the lower pH and at 10 microM concentration of substrate 42 pmol of coproporphyrinogen was formed/min/mg protein. These results compared favorably with those obtained by previously published procedures involving time-consuming extraction and esterification steps.