Porphobilinogen Synthase Activity Research Articles

Copper attenuates early and late biochemical alterations induced by inorganic mercury in young rats

ABSTRACTMercury (Hg), a divalent metal, produces adverse effects predominantly in the renal and central nervous systems. The aim of this study was to determine the effectiveness of copper (Cu) in prevention of mercuric mercury (Hg2+)-mediated toxic effects as well as the role metallothioneins (MT) play in this protective mechanism in young rats. Wistar rats were treated subcutaneously with saline (Sal) or CuCl2.2H2O (Cu 2.6 mg/kg/day) from 3 to 7 days old and with saline or HgCl2 (Hg 3.7 mg/kg/day) from 8 to 12 days old. The experimental groups were (1) Sal-Sal, (2) Cu-Sal, (3) Sal-Hg, and (4) Cu-Hg. MTs and metal contents were determined at 13 and 33 days of age. Porphobilinogen synthase (PBG-synthase) activity as well as renal and hepatic parameters were measured at 33 days. At 13 day, Hg2+ exposure increased hepatic MT, Hg, zinc (Zn) and iron (Fe) levels, in kidney elevated Cu and Hg and decreased renal Fe concentrations, accompanied by elevated blood Hg levels. At 33 days, Hg2+ exposure inhibited renal PBG-synthase activity, increased serum urea levels and lowered Fe and Mg levels. Copper partially prevented the rise in blood Hg and liver Fe noted at 13 days; and completely blocked urea rise and diminished renal PBG-synthase activity inhibition at 33 days. In 13-day-old rats, Cu exposure redistributed the Hg in the body, decreasing hepatic and blood levels while increasing renal levels, accompanied by elevated renal and hepatic MT levels in Hg2+-exposed animals. These results suggest that hepatic MT might bind to hepatic and blood Hg for transport to the kidney in order to be excreted.Abbreviations: MT: metallothioneins; PBG-synthase: porphobilinogen synthase.

Biochemical parameters of pregnant rats and their offspring exposed to different doses of inorganic mercury in drinking water

This work investigated the effects of low and high doses of inorganic mercury in drinking water on biochemical parameters of pregnant rats and their offspring. Female Wistar rats were treated during pregnancy with 0, 0.2, 0.5, 10 or 50μgHg2+/mL as HgCl2. Rats were euthanized on day 20 of pregnancy. Pregnant rats presented a decrease in total water intake in all doses of mercury tested. At high doses, a decrease in the total food intake and in body weight gain was observed. Pregnant rats exposed to 50μgHg2+/mL presented an increase in kidney relative weight. Mercury exposure did not change serum urea and creatinine levels in any of the doses tested. Moreover, mercury exposure did not change porphobilinogen synthase activity of kidney, liver and placenta from pregnant rats in any of the doses tested, whereas fetuses of pregnant rats exposed to 50μgHg2+/mL presented an increase in the hepatic porphobilinogen synthase activity. In general, pregnant rats presented alterations due to HgCl2 exposure in drinking water. However, only the dose 50μgHg2+/mL appeared to be enough to cross the blood-placenta barrier, since at this dose the fetuses presented change in the porphobilinogen synthase activity.

Plasma succinylacetone is persistently raised after liver transplantation in tyrosinaemia type 1

Tyrosinaemia type 1 (HT1) is a rare disorder leading to accumulation of toxic metabolites such as succinylacetone (SA) and a high risk of hepatocellular carcinoma. Children with HT1 traditionally required liver transplantation (OLT) and while the need for this has been reduced by the introduction of nitisinone some still require OLT. SA inhibits the enzyme porphobilinogen (PBG) synthase and its activity can be used as a marker of active SA. Elevated urinary SA post OLT has been reported previously. This study describes a novel finding of elevated plasma SA following OLT for HT1. A retrospective analysis was performed of patients treated for HT1 at our institution from 1989-2010. Thirteen patients had an OLT for HT1. In patients who received nitisinone prior to OLT, mean urinary and plasma SA were elevated prior to treatment but normalised by the time of OLT (p ≤ 0.01). Mean PBG synthase activity increased from 0.032 to 0.99 nkat/gHb (ref range 0.58-1.25) at the time of OLT (p < 0.01). Mean urinary SA in patients not treated with nitisinone was also elevated prior to OLT; plasma levels and PBG synthase activity were not available prior to OLT for this group. Following OLT, mean urinary and plasma SA were elevated in all for the duration of follow-up and associated with low-normal PBG synthase activity. Urinary and plasma SA levels are elevated following OLT for HT1. Low-normal PBG synthase activity suggests the plasma SA may be active. The clinical significance of this is unclear.

P79 Plasma succinylacetone is raised after liver transplantation for tyrosinaemia type 1 and is associated with reduced porphobilinogen synthase activity suggesting it is functional

IntroductionTyrosinaemia type 1 (TT1) is a rare disorder of tyrosine metabolism leading to accumulation of toxic metabolites such as succinylacetone (SA) and a high risk of hepatocellular carcinoma. Children with...

Delayed biochemical changes induced by mercury intoxication are prevented by zinc pre-exposure

This work evaluated the delayed effects of mercury and the effectiveness of zinc in preventing such effects. Pups were pre-treated with 1 daily dose of ZnCl2 (27mg/kg/day, by subcutaneous injections) from 3rd to 7th postnatal day and received 1 daily dose of 5mg/kg of HgCl2, for 5 subsequent days (8–12 days old). Animals were euthanized 21 days after the end of Hg-exposure. Porphobilinogen-synthase activity as well as zinc and mercury contents was determined in the liver and kidneys. Alanine aminotransferase, aspartate aminotransferase and lactic dehydrogenase activities as well as urea, creatinine and glucose levels were analyzed in plasma or serum. Some animals were considered more sensitive to mercury, since they did not recover the body weight gain and presented an increase of renal and hepatic mercury content, urea and creatinine levels; a decrease in renal porphobilinogen-synthase and alanine aminotransferase activities, as well as a decrease in the liver and an increase in kidney weights. Some animals were considered less sensitive to mercury because they recovered the body weight and presented no biochemical alterations in spite of mercury in the tissues. Zinc prevents partially or totally the alterations caused by mercury even those that persisted for a long time after the end of exposure. These findings suggest that there is difference among the animals regarding the sensitivity to mercury.

Pseudomonas aeruginosa porphobilinogen synthase assembly state regulators: hit discovery and initial SAR studies

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of the essential heme, chlorophyll and vitamin B(12) heme pigments. PBGS activity is regulated by assembly state, with certain oligomers exhibiting biological activity and others either partially or completely inactive, affording an innovative means of allosteric drug action. Pseudomonas aeruginosa PBGS is functionally active as an octamer, and inactive as a dimer. We have identified a series of compounds that stabilize the inactive P. aeruginosa dimer by a computational prescreen followed by native PAGE gel mobility shift analysis. From those results, we have prepared related thiadiazoles and evaluated their ability to regulate P. aeruginosa PBGS assembly state.

Allosteric Inhibition of Human Porphobilinogen Synthase

Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329-337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of approximately 111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90-100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme.

Determination of EDTA in dried blood samples by tandem mass spectrometry avoids serious errors in newborn screening

Newborn screening programs use whole blood dried on filter paper as the standard specimen. Metabolites are reasonably stable and can easily be sent to screening laboratories by regular mail. The recommended sample collection procedure is to spot native blood without anticoagulants onto the filter paper, because anticoagulants can interfere with the different laboratory methods. However, visual examination of the blood spots cannot always detect contamination. In this study, whole blood was drawn by venous puncture from a healthy volunteer, spiked with the corresponding metabolites and EDTA, and spotted onto filter paper. TSH and 17alpha-hydroxyprogesterone were determined by time resolved fluoroimmunoassays with the AutoDelfia system. Total galactose, biotinidase activity, and galactose-1-phosphate uridyltransferase activity were measured photometrically or fluorometrically. Succinyl acetone was estimated indirectly through the inhibition of porphobilinogen synthase activity (PBGS assay). EDTA, amino acids, and acylcarnitines were converted to the corresponding butyl esters, after extraction with methanol, and analysed by LC-MS/MS. EDTA contamination gives falsely elevated 17-OHP values and falsely reduced TSH and PBGS values. The inclusion of an EDTA determination in routine screening revealed that at least 0.06% of newborn screening samples were contaminated with EDTA. In conclusion, non-conformity during the pre-analytical phase is a source of false positive and false negative screening results. Determination of EDTA from NBS blood spots can reliably identify these samples and prevent screening errors.

ALAD Porphyria Is a Conformational Disease

ALAD porphyria is a rare porphyric disorder, with five documented compound heterozygous patients, and it is caused by a profound lack of porphobilinogen synthase (PBGS) activity. PBGS, also called "delta-aminolevulinate dehydratase," is encoded by the ALAD gene and catalyzes the second step in the biosynthesis of heme. ALAD porphyria is a recessive disorder; there are two common variant ALAD alleles, which encode K59 and N59, and eight known porphyria-associated ALAD mutations, which encode F12L, E89K, C132R, G133R, V153M, R240W, A274T, and V275M. Human PBGS exists as an equilibrium of functionally distinct quaternary structure assemblies, known as "morpheeins," in which one functional homo-oligomer can dissociate, change conformation, and reassociate into a different oligomer. In the case of human PBGS, the two assemblies are a high-activity octamer and a low-activity hexamer. The current study quantifies the morpheein forms of human PBGS for the common and porphyria-associated variants. Heterologous expression in Escherichia coli, followed by separation of the octameric and hexameric assemblies on an ion-exchange column, showed that the percentage of hexamer for F12L (100%), R240W (80%), G133R (48%), C132R (36%), E89K (31%), and A274T (14%) was appreciably larger than for the wild-type proteins K59 and N59 (0% and 3%, respectively). All eight porphyria-associated variants, including V153M and V275M, showed an increased propensity to form the hexamer, according to a kinetic analysis. Thus, all porphyria-associated human PBGS variants are found to shift the morpheein equilibrium for PBGS toward the less active hexamer. We propose that the disequilibrium of morpheein assemblies broadens the definition of conformational diseases beyond the prion disorders and that ALAD porphyria is the first example of a morpheein-based conformational disease.

2,3‐Dimercapto‐1‐propanol does Not Alter the Porphobilinogen Synthase Inhibition but Decreases the Mercury Content in Liver and Kidney of Suckling Rats Exposed to HgCl2

Heavy metals have received great attention as environmental pollutants mainly because once introduced in the biological cycle they are incorporated in the food chain. Especially the mercury toxicity due to a diversity of effects caused by different chemical species should be emphasized. Heavy metal intoxication has been treated with chelating agents such as 2,3-dimercapto-1-propanol (BAL). However, the efficacy of this treatment is questionable due to the lack of specific effect on the toxic metal. The present study examined the effects of HgCl2 exposure (five doses of 5.0 mg/kg between ages 8 to 12 days) on physiological parameters, on porphobilinogen synthase activity, and on mercury content in liver, kidneys and brain from suckling rats. The effect of BAL (one dose of 12.5-75 mg/kg) applied 24 hr after mercury intoxication on these parameters was also investigated. The results demonstrate that HgCl2 intoxication induced a decrease of corporal weight gain as well as brain weight and an increase in renal weight. The inhibition of porphobilinogen synthase from liver and kidney, is still significant and was not modified by subsequent BAL treatment. However, BAL altered two effects induced by mercury: increase in death percentage and decrease in mercury contents in liver and kidney. The increase of mortality induced by mercury was not promoted by metal redistribution to brain nor by the increase of porphobilinogen synthase inhibition induced by metal. More investigations are necessary to determine if the different effects of BAL on intoxication by metals are possibly related to other tissues and/or if the probable metal-chelating complex formed is more toxic than the metal itself.

Developmental profiles of 5-aminolevulinate, porphobilinogen and porphobilinogen synthase activity in Pieris brassicae related to the synthesis of the bilin-binding protein

The bilin-binding protein (BBP) occurs as a major soluble protein in haemolymph, fat body, epidermis and wings of Pieris brassicae. It is a member of the lipocalin protein superfamily with yet unknown function. Here, we studied the developmental regulation of tetrapyrrole biosynthesis that provides the bilin ligand as the predominating end product. The levels of the precursors 5-aminolevulinate (ALA) and porphobilinogen (PBG) varied during larval–pupal transition in accordance with the activity of the related enzyme porphobilinogen synthase (PBGS). During adult development, both precursors were low while PBGS activity increased parallel to the formation of BBP, as shown in previous work. A competitive inhibitor of PBGS was partially purified from the meconium and characterised as a heat-stabile acidic compound. Label from [ 14C]ALA, injected into developing pupae of different age, was found to 80% in the hind wings and to 20% in the forewings after adult eclosion, reflecting the unequal distribution of BBP between the pairs of wings. This contrasted to the activity of PBGS that was equally active in forewings and hind wings. Together with the variation of enzyme activity during wing development our results led us propose that the (hind) wings may play a role in the synthesis of the tetrapyrrole ligand of BBP.

Effect of Group 13 metals on porphobilinogen synthase in vitro

Mammalian porphobilinogen synthase (PBGS) is a metalloenzyme, which requires Zn 2+ and reduced thiol groups for maximal catalytic activity, and is an important molecular target for the widespread environmental toxic metals. The mechanism underlying the PBGS inhibition by elements of Group 13 metals (Al 3+, Ga 3+, In 3+, and Tl 3+) has not yet been determined. The main objective of the present study was to characterize, in a comparative way, the molecular mechanism of PBGS inhibition caused by salts of elements of Group 13. Al 3+, Ga 3+, and In 3+ i inhibited purified hepatic bovine PBGS, and the IC 50 for PBGS inhibition by Ga 3+ (IC 50 = 442 ± 63 μmol l −1) was higher than that for Al 3+ (IC 50 = 319 ± 41 μmol l −1) and In 3+ (IC 50 = 298 ± 44 μmol l −1). Zn 2+ restored completely aluminum-induced inhibitory effect on PBGS activity. Tl 3+ inhibited liver bovine PBGS (IC 50 = 8.5 ± 0.9 μmol l −1) and glutathione reduced markedly this inhibitory effect (IC 50 = 87 ± 8 μmol l −1). GSH had no protective effects on the inhibitory actions of Al 3+ and Ga 3+ against PBGS; in contrast, GSH reduced the inhibitory effect of In 3+ on PBGS. dl-Dithiothreitol (DTT) restored completely the enzyme activity inhibited by Tl 3+ and had only a modest effect on the inhibitory effect of In 3+. Zn 2+ was unable to change the inhibitory effect of Tl 3+ on liver bovine PBGS; in contrast, Zn 2+ recovered almost completely the enzyme inhibition caused by In 3+ and Ga 3+. Thus, our results indicated that Al 3+, Ga 3+, and In 3+ inhibit PBGS by competing with Zn 2+, whereas Tl 3+ and In 3+ inhibit bovine PBGS by directly oxidizing essential sulfhydryl groups.

Effects of Different Concentrations and Form Ratios of Nitrogen on Chlorophyll Biosynthesis,Photosynthesis, and Biomass Partitioning in Fraxinus mandshurica Seedlings

Fraxinus mandshurica, native to northeastern China, is an important timber tree. The tree often suffers from nitrogen (N) deficiency because long winter results in slow decomposition rate of litter. In the present study, effects of different concentrations and form ratios of nitrogen on chlorophyll biosynthesis, total leaf N, soluble protein, and photosynthesis were studied. During the N concentration treatment, biosynthesis rate of 5_aminolevulinic acid (ALA) and activity of porphobilinogen (PBG) synthase showed irregular changes, but total chlorophyll content increased when N concentrations varied from 1 to 8 mmol·L -1, and activity of PBG synthase did not change significantly. Total leaf N, soluble leaf proteins, and photosynthesis rate also increased when N concentrations increased from 1 to 8 mmol·L -1. These results suggested that N concentrations did not affect biosynthesis rates of ALA and activities of PBG synthase and 8 mmol·L -1 of N most increased total leaf N, soluble proteins in leaves, and photosynthesis rate. During the N form treatment, biosynthesis of ALA increased when the ratio of NO - 3_N increased from 0 to 50%, but activity of PBG synthase, total chlorophyll concentrations, total leaf N and soluble leaf proteins increased when the ratio of NO - 3_N increased from 0 to 75%. Photosynthesis rate also increased when the ratio of NO - 3_N increased from 0 to 75%. During the two types of treatments, there were significant relationships between total leaf N, soluble protein concentrations and net photosynthesis rates. These results suggested that F. mandshurica seedlings showed a preference for NO - 3_N, but the ratio of 75% NO - 3_N to 25% NH + 4_N promoted an increase in chlorophyll biosynthesis, accumulation of N and soluble proteins in leaves, and net photosynthesis rate. Total biomass increased as exogenous nitrogen concentrations increased from 1 to 8 mmol·L -1. Biomass partitioning to roots decreased, and biomass partitioning to shoots increased. As ratios of NO - 3_N to NH + 4_N increased from 0 to 75%, total biomass increased, but biomass partitioning to roots increased as ratios of NO - 3_N to NH + 4_N increased from 0 to 100%. When both nitrogen concentration treatment and form ratio treatment were carried out, biomass accumulation was closely related to photosynthesis rate, and the relationship between biomass accumulation and photosynthesis was related to nitrogen partitioning in leaves. It is proposed that 8 mmol·L -1 and 75% NO - 3_N∶25%NH + 4_N should be used in practical cultivation of F. mandshurica seedlings.

Oxygen-mediated regulation of porphobilinogen formation in Rhodobacter capsulatus.

A Rhodobacter capsulatus hemC mutant has been isolated and used to show that oxygen regulates the intracellular levels of porphobilinogen. Experiments using a hemB-cat gene fusion demonstrated that oxygen does not transcriptionally regulate hemB transcription. Porphobilinogen synthase activity is not regulated by oxygen nor is the enzyme feedback inhibited by hemin or protoporphyrin IX. It was demonstrated that less than 20% of [(14)C]aminolevulinate was incorporated into bacteriochlorophyll, suggesting that the majority of the aminolevulinate is diverted from the common tetrapyrrole pathway. Porphobilinogen oxygenase activity was not observed in this organism; however, an NADPH-linked aminolevulinate dehydrogenase activity was demonstrated. The specific activity of this enzyme increased with increasing oxygen tension. The results presented here suggest that carbon flow over the common tetrapyrrole pathway is regulated by a combination of feedback inhibition of aminolevulinate synthase and diversion of aminolevulinate from the pathway by aminolevulinate dehydrogenase.

Alcohol and porphyrin metabolism.

Alcohol is a porphyrinogenic agent which may cause disturbances in porphyrin metabolism in healthy persons as well as biochemical and clinical manifestations of acute and chronic hepatic porphyrias. After excessive consumption of alcohol, a temporary, clinically asymptomatic secondary hepatic coproporphyrinuria is observable, which can become persistent in cases of alcohol-induced liver damage. Nowadays, the alcohol-liver-porphyrinuria syndrome is the first to be mentioned in secondary hepatic disturbances of porphyrin metabolism. Acute hepatic porphyrias (acute intermittent porphyria, variegate porphyria and hereditary coproporphyria) are considered to be molecular regulatory diseases, in contrast to non-acute, chronic hepatic porphyria, clinically appearing as porphyria cutanea tarda (PCT). Porphyrins do not accumulate in the liver in acute porphyrias, whereas in chronic hepatic porphyrias they do. Thus, chronic hepatic porphyria is a porphyrin-accumulation disease, whereas acute hepatic porphyrias are haem-pathway-dysregulation diseases, characterized in general by induction of delta-aminolevulinic acid synthase in the liver and excessive stimulation of the pathway without storage of porphyrins in the liver. The clinical expression of acute hepatic porphyrias can be triggered by alcohol, because alcohol augments the inducibility of delta-aminolevulinic acid synthase. In chronic hepatic porphyrias, however, which are already associated with liver damage, alcohol potentiates the disturbance of the decarboxylation of uro- and heptacarboxyporphyrinogen, which is followed by a hepatic accumulation of uro- and heptacarboxyporphyrin and their sometimes extreme urinary excretion. Especially in persons with a genetic deficiency of uroporphyrinogen decarboxylase, but also in patients with the so-called sporadic variety of PCT, alcohol is able to transform an asymptomatic coproporphyrinuria into PCT. Alcohol has many biochemical and clinical effects on porphyrin and haem synthesis both in humans and laboratory animals. Ethanol suppresses the activity of porphobilinogen synthase (synonym: delta-aminolevulinic acid dehydratase), uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and ferrochelatase, whereas it induces the first and rate-limiting enzyme in the pathway, delta-aminolevulinic acid synthase and also porphobilinogen deaminase. Therefore, teetotalism is a therapeutically and prophylactically important measure in all types of hepatic porphyrias.

A role for HEM2 in cadmium tolerance

A Candida glabrata cadmium-sensitive mutant partially defective in glutathione production and exhibiting a complete absence of phytochelatins was used to clone a gene required for Cd tolerance. Transformation of the Cd-sensitive mutant with a genomic library from the wild-type C. glabrata led to the cloning of a gene that restored Cd tolerance and formation of Cd-glutathione and Cd-phytochelatin complexes. The cloned gene showed high levels of nucleic acid and protein sequence homology to the HEM2 genes, encoding porphobilinogen synthases, from several sources. It was shown that the C. glabrata Cd-sensitive mutant indeed exhibited a significant reduction in porphobilinogen synthase levels. The cloned C. glabrata gene complemented a hem2 mutant of Saccharomyces cerevisiae and restored porphobilinogen synthase activity in the mutant. The Cd sensitive mutant predictably showed decreased levels of sulfite reductase that requires siroheme, a metabolite produced in the heme biosynthetic pathway. The addition of cysteine, but not methionine, increased glutathione levels and Cd tolerance of both the wild-type and the mutant strain. However, addition of hemin chloride and methionine together restored Cd tolerance indicating that heme was required for transsulfuration of homocysteine to cysteine.

Structure and Expression of the Chlorobium vibrioforme hemB Gene and Characterization of its Encoded Enzyme, Porphobilinogen Synthase

Plasmids containing DNA from the green photosynthetic bacterium Chlorobium vibrioforme complement a heme-requiring Escherichia coli hemB mutant that is deficient in porphobilinogen (PBG) synthase activity. PBG synthase activity was detected in extract of complemented cells but not in that of cells transformed with control plasmid. The sequence of the C. vibrioforme hemB gene predicts a HemB protein that contains 328 amino acids, has a molecular weight of 36,407, and is 53% identical to the homologous proteins of Synechocystis sp. PCC 6301 and Rhodobacter capsulatus. The response of C. vibrioforme PBG synthase to divalent metals is unlike that of any previously described PBG synthase; Mg2+ stimulates but is not required for activity, and Zn2+ neither stimulates nor is required. This response correlates with predicted sequences of two putative variable metal binding regions of C. vibrioforme HemB. The C. vibrioforme hemB open reading frame begins 1585 bases downstream from the end of the hemD open reading frame and is transcribed in the same direction as hemA, hemC, and hemD. However, hemB is not part of the same transcription unit as these genes, and the hemB transcript is approximately the same size as the hemB gene alone. Between hemD and hemB there is an intervening open reading frame that is oriented in the opposite direction and encodes a protein with a predicted amino acid sequence significantly similar to that of inositol monophosphatase, an enzyme that is not involved in tetrapyrrole biosynthesis. The gene order within hem gene clusters is highly conserved in phylogenetically diverse prokaryotic organisms. This conservation suggests that there are functional constraints on the relative order of the hem genes.

The affinity of human erythrocyte porphobilinogen synthase for Zn2+ and Pb2+.

Porphobilinogen synthase activity has been measured in human erythrocyte lysates supplemented with metal-ion buffers to control free Zn2+ and Pb2+ concentrations. The enzyme is activated by Zn2+ with a Km of 1.6 pM and inhibited by Pb2+ with a Ki of 0.07 pM. Pb2+ and Zn2+ appear to compete for a single metal-binding site. The half-time for loss of Zn2+ from the active site, or replacement of Pb2+ by Zn2+, were in the 10-20-min range at 37 degrees C. Zn2+ did not affect the affinity for the substrate 5-aminolevulinate, but Pb2+ reduced it non-competitively. All the experiments were conducted with a blood sample of the common 1-1 phenotype [Astrin, K. H., Bishop, D. F., Wetmur, J. G., Kaul, B., Davidow, B. & Desnick, R. J. (1987) Ann. NY Acad. Sci. 514, 23-29].

Porphobilinogen Synthase from Escherichia coli is a Zn(II) Metalloenzyme Stimulated by Mg(II)

Porphobilinogen synthase (PBGS) is essential to all life forms; in mammals it is definitively established that Zn(II) is required for activity. The literature regarding the metal requirement for PBGS in other animals, plants, and bacteria neither establishes nor disproves a Zn(II) requirement. We have characterized Escherichia coli PBGS and found it to be remarkably similar to bovine PBGS. The similarities include a requirement for Zn(II), inhibition by 1,10-phenanthroline, an exceptional thermal stability, a requirement for free sulfhydryl(s) as shown by sensitivity to modification by methyl methanethiosulfonate, and the presence of tightly bound product on freshly isolated enzyme. Proton-induced X-ray emission analysis shows E. coli PBGS to contain a stoichiometric amount of Zn and no other metals. The most striking similarity between E. coli and bovine PBGS is the 13C NMR spectrum of enzyme-bound [3,5-13C]PBG; the chemical shifts of bound product are identical for both bovine and E. coli PBGS. Minor differences between E. coli PBGS and its mammalian counterpart include Km (ALA) = 1.9 mM, a pH optimum of 7.5-8, and its molar absorbtion coefficient expressed as A0.1%280 is 0.588. We conclude from these data that E. coli PBGS is a Zn(II)-metalloenzyme and that Zn(II) is required for catalytic activity, and propose that the mammalian and bacterial PBGS function by similar mechanisms. There is one significant difference between E. coli and mammalian PBGS. For E. coli PeGS, Mg(II) causes a twofold stimulation of the Zn(II)-induced E. coli PBGS activity; this effect is not seen for bovine PBGS. The stimulation of activity by Mg(II) mimics the effect of Mg(II) on plant PBGS, although E. coli PBGS does not contain the putative Mg(II) binding site recently revealed by Boese et al. [Q. F. Boese, A. J. Spano, T. Li, and M. P. Timko (1991) J. Biol. Chem. 266, 17060-170661. This work lays the foundation for identification of functional amino acids based on the sequence similarities between PBGS from bacterial, plant, and mammalian sources.

Effects of chronic exposure to sublethal concentrations of lead acetate on heme synthesis and immune function in red-tailed hawks

Red-tailed hawks were exposed to sublethal levels of lead acetate for periods of 3 or 11 weeks. Alterations in the heme biosynthetic pathway were demonstrated after the first week of exposure to 0.82 mg lead per kilogram body weight per day. Activity of erythrocyte porphobilinogen synthase (aminolevulinic acid dehydratase) was depressed significantly and did not return to normal levels until 5 weeks after the termination of lead treatments. A rapid and relatively brief increase in erythrocyte free protoporphyrin and a slower but more prolonged increase in its zinc complex were also demonstrated with exposure to this dose of lead for 3 weeks. Less substantial decreases in hematocrit and hemoglobin levels occurred but only in the longer experiment with exposure to higher lead levels. Short term, low level lead exposure did not effect immune function significantly in the hawks, as measured by antibody titers to foreign red blood cells or by the mitogenic stimulation of T-lymphocytes. Increased lead exposure produced a significant decrease in the mitogenic response but had no effect on antibody titers.