Regulation of rat liver tryptophan pyrrolase by its cofactor haem: Experiments with haematin and 5-aminolaevulinate and comparison with the substrate and hormonal mechanisms.

1. The administration of haematin or 5-aminolaevulinate to rat enhances the activity of liver tryptophan pyrrolase; both endogenous and newly formed apoenzymes become strongly haem-saturated. Haem activation does not stabilize tryptophan pyrrolase. 2. Actinomycin D, puromycin or cycloheximide prevent the activation of the enzyme by 5-aminolaevulinate but not that by haematin. The latter is inhibited by haem-destroying porphyrogens. 3. The combined injection of either haematin or 5-aminolaevulinate with cortisol does not produce an additive effect, whereas potentation is observed when tryptophan is jointly given with either the cofactor or the haem precursor. 4. Further experiments on the substrate (tryptophan) mechanism of pyrrolase regulation are reported, and a comparison between this and the cofactor and hormonal mechanisms is made. 5. It is suggested that the substrate mechanism may also involve increased haem synthesis. 6. The role of tryptophan pyrrolase in the utilization of liver haem, and as a possible model for the exacerbation by drugs of human hepatic porphyrias, is discussed.     

Tryptophan pyrrolase in haem regulation. The relationship between the depletion of rat liver tryptophan pyrrolase haem and the enhancement of 5-aminolaevulinate synthase activity by 2-allyl-2-isopropylacetamide.

Rat liver tryptophan pyrrolase haem is maximally depleted at 30 min after administration of a 400 mg/kg dose of 2-allyl-2-isopropylacetamide. This depletion lasts for 24 h, by which time 5-aminoleevulinate synthase activity becomes maximally enhanced. 2. though the above maximum depletion of pyrrolase haem (at 0.5h) is also produced by a 100 mg/kg dose of the porphyrogen, this does not enhance synthase activity at 24 h. It and smaller doses, however, cause a smaller but earlier enhancement of synthase activity (maximum at 2 h) and produce a similarly short-lived deplation of pyrrolase haem. 3. The depletion of pyrrolase haem and the enhancement of synthase activity by the porphyrogen are inhibited by compound SKF 525-A and phenazine methosulphate, and are potentiated by nicotinamide but not by phenobarbitone. Phenazine methosulphate and nicotinamide also exert opposite effects on hexobarbital sleeping-time. 4. 2-Allyl-2-isopropylacetamde also the depletes pyrrolase haem in vitro. It does so in liver homogenates of control rats in the presence, and in those of phenobarbitone-treated rats in the absence of added NADPH. 5. A discussion of the present results in relation to previous work with other haemoproteins suggests that, whereas cytochrome P-450 (haem) is primarily involved in the production of the active (porphyrogenic) metabolite(s) of 2-allyl-2-isopropylacetamide, the haem pool used by tryptophan pyrrolase may play an important role in the effects of this compound on haem biosynthesis.     

Tryptophan pyrrolase in haem regulation. The mechanism of the permissive effect of cortisol on the enhancement of 5-aminolaevulinate synthase activity by 2-allyl-2-isopropylacetamide in the adrenalectomized-rat liver.

The decreased ability of 2-allyl-2-isopropropylacetamide to enhance liver 5-amino-laevulinate synthase activity in the adrenalectomized rat is not associated with a marked depletion of the already low amount of tryptophan pyrrolase haem. Cortisol permits the porphyrogen markedly to enhance synthase activity by rendering it capable of causing a stronger depletion of pyrrolase haem, presumably as result of hormonal induction of pyrrolase synthesis.     

Tryptophan pyrrolase in haem regulation. Experiments with administered haematin and the relationship between the haem saturation of tryptophan pyrrolase and the activity of 5-aminolaevulinate synthase in rat liver.

1. Administration of haematin to rats decreases 5-aminolaevulinate synthase activity in whole liver homogenates. 2. An inverse relationship between this decrease and the increase in saturation of apo-(tryptophan pyrrolase) with haem is observed during the initial phase of treatment with haematin. 3. Significant changes in both functions are caused by a 1 mg/kg dose of haematin, whereas the maximum effects are achieved by the 5 mg/kg dose. 4. Prevention by allopurinol of the conjugation of exogenously administered haematin with apo-(tryptophan pyrrolase) renders this haem available for further repression of 5 aminolaevulinate synthase. 5. The various aspects of the relationship between synthase activity and the haem saturation of tryptophan pyrrolase are discussed.     

The effects of chemical porphyrogens and drugs on the activity of rat liver tryptophan pyrrolase

1. Drugs such as phenobarbitone and phenylbutazone, which increase the concentration of microsomal haem and cytochrome P-450, also increase the saturation of rat liver apo-(tryptophan pyrrolase) with its haem activator, as does the haem precursor 5-aminolaevulinate. 2. At 4h after the administration of the porphyrogens 2-allyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydrocollidine and griseofulvin, the total pyrrolase activity is increased whereas the haem saturation of the apoenzyme is decreased. This decreased saturation is prevented by pretreatment of the animals with the inhibitor of drug-metabolizing enzymes, SKF 525-A. 3. Pretreatment of rats with the above porphyrogens inhibits the rise in holo-(tryptophan pyrrolase) activity produced by subsequent administration of cortisol, tryptophan and 5-aminolaevulinate with two single exceptions, the possible reasons for which are discussed. 4. At 24h after the administration, in starved rats, of a single daily injection of the above porphyrogens for 1 or 2 days, the holoenzyme activity is significantly increased. 5. It is suggested that the saturation of rat liver apo-(tryptophan pyrrolase) with its haem activator can be modified by treatment known to cause destruction, inhibition of synthesis, increased utilization and enhanced synthesis of liver haem. The possible involvement of the latter phenomenon in the aetiology of mental disorders in some patients with porphyria is discussed.     

Tryptophan and tryptophan pyrrolase in haem regulation. The role of lipolysis and direct displacement of serum-protein-bound tryptophan in the opposite effects of administration of endotoxin, morphine, palmitate, salicylate and theophylline on rat liver 5-aminolaevulinate synthase activity and the haem saturation of tryptophan pyrrolase

1. The increase in the haem saturation of rat liver tryptophan pyrrolase caused by tryptophan administration was previously shown to be associated with a decrease in 5-aminolaevulinate synthase activity. 2. It is now shown that similar reciprocal effects are caused by palmitate and salicylate, both of which increase tryptophan availability to the liver by direct displacement of the serum-protein-bound amino acid. 3. The reciprocal effects on the former two parameters caused by endotoxin and morphine are associated with an increase in liver tryptophan concentration produced by a lipolysis-dependent, non-esterified fatty acid-mediated, displacement of the serum-protein-bound amino acid. 4. All these changes and those caused by another lipolytic agent, theophylline, are prevented by the β-adrenoceptor-blocking agent propranolol and by the opiate-receptor antagonist naloxone, whose anti-lipolytic nature is demonstrated. 5. High correlation coefficients have been obtained for one or more pairs of the following parameters: serum non-esterified fatty acid concentration, free serum tryptophan concentration, liver tryptophan concentration, liver 5-aminolaevulinate synthase activity, liver holo-(tryptophan pyrrolase) activity and the haem saturation of liver tryptophan pyrrolase. 6. It is suggested that liver tryptophan concentration may play an important role in the regulation of 5-aminolaevulinate synthase synthesis, and that the latter may be subject to control by changes in lipid metabolism and may be influenced by pharmacological agents that affect tryptophan disposition. 7. Preliminary evidence suggests that tryptophan may be bound in the liver and that such a possible binding may control its availability for its hepatic functions.     

The functions and regulation of tryptophan pyrrolase.

The regulation and functions of rat liver tryptophan pyrrolase are reviewed. The enzyme is regulated by four mechanisms: hormonal induction by glucocorticoids, substrate activation and stabilization by tryptophan, cofactor activation by haem and feedback inhibition by NAD(P)H. Possible functions of the pyrrolase are the detoxication of tryptophan and the regulation of brain 5-hydroxytryptamine synthesis, liver haem metabolism, synthesis of nicotinamide-adenine dinucleotides (phosphates) and hepatic gluconeogenesis.It is suggested that the regulation and functions of tryptophan pyrrolase are physiologically interrelated, and that the enzyme may play important roles in vital body processes.     

Tryptophan pyrrolase in haem regulation. The mechanisms of enhancement of rat liver 5-aminolaevulinate synthase activity by starvation and of the glucose effect on induction of the enzyme by 2-allyl-2-isopropylacetamide

1. Rat liver tryptophan pyrrolase activity is enhanced by a hormonal-type mechanism during the first 2 days of starvation and by a substrate-type mechanism during the subsequent 2 days. 5-Aminolaevulinate synthase activity is also enhanced during the first 2 days of starvation, but returns thereafter to values resembling those observed in the fed rat. Treatments that prevent or reversé the enhancement of tryptophan pyrrolase activity in 24–48h-starved rats also abolish that of 5-aminolaevulinate synthase activity. Starvation of guinea pigs, which does not enhance the pyrrolase activity, also fails to alter that of the synthase. It is suggested that the decrease in 5-aminolaevulinate synthase activity in 72–96h-starved rats represents negative-feedback repression of synthesis, possibly involving tryptophan participation, whereas the enhancement observed in 24–48h-starved animals is caused by positive-feedback induction secondarily to increased utilization of the regulatory-haem pool by the newly synthesized apo-(tryptophan pyrrolase). 2. Glucose, fructose and sucrose abolish the 24h-starvation-induced increases in rat liver tryptophan pyrrolase and 5-aminolaevulinate synthase activities. Cortisol reverses the glucose effect on 5-aminolaevulinate synthase activity, presumably by enabling pyrrolase to re-utilize the regulatory-haem pool after induction of synthesis of this latter enzyme. 3. The impaired ability of 2-allyl-2-isopropylacetamide to enhance markedly 5-aminolaevulinate synthase activity in 24h-starved rats treated with glucose is associated with a failure of the porphyrogen to cause loss of tryptophan pyrrolase haem. Cortisol restores the ability of the porphyrogen to destroy tryptophan pyrrolase haem and to enhance markedly 5-aminolaevulinate synthase activity, presumably by enhancing tryptophan pyrrolase synthesis and, thereby, its re-utilization of the regulatory-haem pool. It is tentatively suggested that 2-allyl-2-isopropylacetamide destroys the above pool only after it has become bound to (or utilized by) apo-(tryptophan pyrrolase).     

The effects of acetate, metal cations, phenobarbitone, porphyrogens and substrates of glycine acyltransferase on the utilization of haem by rat liver apo-(tryptophan pyrrolase).

1. The utilization of haem by rat liver apo-(tryptophan pyrrolase) under basal conditions and after enhancement of the enzyme activity by various mechanisms was studied under the influence of treatments affecting various aspects of liver haem metabolism. 2. These treatments were: benzoate and p-aminobenzoate as substrates of glycine acyltransferase, acetate as an inhibitor of 5-aminolaevulinate synthase activity, enhancement of 5-aminolaevulinate dehydratase by aluminium, destruction of haem and inhibition of ferrochelatase by porphyrogens, increased haem utilization by phenobarbitone and enhancement of haem oxygenase activity by metal cations. 3. The results show that the haem saturation of the apoenzyme is sensitive to all these treatments. 4. The possible usefulness of tryptophan pyrrolase in studying the regulation of liver haem is suggested.     

Effects of the haem precursor 5-aminolaevulinate on tryptophan metabolism and disposition in the rat.

5-Aminolaevulinate administration to rats inhibits cerebral 5-hydroxytryptamine synthesis by decreasing tryptophan availability to the brain secondarily to activation of hepatic tryptophan pyrrolase. The results show that tryptophan metabolism and disposition can be influenced by changes in liver haem concentration, and are discussed briefly in relation to mood disorders in the hepatic porphyrias.     

Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450.

After its administration to intact rats, haemoglobin haem was incorporated into hepatic tryptophan pyrrolase as shown by the marked increase in functional constitution of this enzyme. Incorporation of haemoglobin haem into cytochrome P-450 was demonstrated in intact rats and in the isolated rat liver perfused with haemoglobin-free medium. In both systems, haemoglobin haem restored cytochrome P-450 content and its dependent mixed-function-oxidase activity after substrate-induced destruction of the cytochrome P-450 haem moiety. Further confirmation that haemoglobin haem could be incorporated prosthetically into cytochrome P-450 was achieved by administration of [3H]haemoglobin to rats and subsequent isolation and characterization of radiolabelled substrate-alkylated products of cytochrome P-450 haem. Our findings indicate that, although hepatic uptake of parenteral haemoglobin is slower than that of haem, it appears to serve as an effective haem donor to the intrahepatic 'free' haem pool. Thus parenteral haemoglobin may warrant consideration as a therapeutic alternative to haem in the acute hepatic porphyrias.     

The mechanism of inhibition of rat liver tryptophan pyrrolase activity by 4-hydroxypyrazolo[3,4-d]pyrimidine (allopurinol)

1. Allopurinol (4-hydroxypyrazolo[3,4-d]pyrimidine) selectively inhibits the apotryptophan pyrrolase activity in homogenates of rat liver in vitro and after intraperitoneal administration. The inhibition is abolished by an excess of haematin. The allopurinol metabolite alloxanthine has no effect on the pyrrolase activity in vitro or after administration. Allopurinol also inhibits the activation of the enzyme in vitro by ascorbate, ethanol plus NAD(+), NADH, hypoxanthine or xanthine. It is suggested that these agents cause the conversion of a latent form of the pyrrolase into the apoenzyme, and that xanthine oxidase is not involved in this process. 2. The raised total pyrrolase activity observed after the administration of cortisol, cyclic AMP, tryptophan, salicylate or ethanol is lowered by allopurinol in vitro to the corresponding holoenzyme values. A similar effect is observed when allopurinol is administered shortly before cortisol or cyclic AMP. Pretreatment of rats with allopurinol completely prevents the enhancement of the pyrrolase activities by tryptophan, salicylate or ethanol. 3. It is suggested that allopurinol inhibits rat liver tryptophan pyrrolase activity in vitro and after administration by preventing the conjugation of the apoenzyme with its haem activator. The possible usefulness of combined allopurinol-tryptophan therapy of affective disorders is discussed.     

Evidence that in chick embryos destruction of hepatic microsomal cytochrome P-450 haem is a general mechanism of induction of delta-aminolaevulinate synthase by porphyria-causing drugs.

A variety of prophyrinogenic compounds were tested for their effect in ovo on chick-embryo liver microsomal cytochrome P-450 haem concentration and mitochondrial delta-aminolaevulinate synthase activity. With all drugs tested, there was a 30--50% decrease in cytochrome P-450 haem concentration within 1 h of treatment, and this was closely followed by an increase in delta-aminolaevulinate synthase activity. The relationship was independent of the extent of enzyme induction and is consistent with the proposal that drug-mediated destruction of cytochrome P-450 haem is the primary mechanism of induction of delta-aminolaevulinate synthase. After induction, synthesis of delta-aminolaevulinate synthase could be maintained by inhibiting further haem synthesis. These studies suggest that induction of porphyria is a combination of two distinct processes: (a) induction of delta-aminolaevulinate synthase synthesis by destruction of cytochrome P-450 haem and consequent depletion of cellular free haem; (b) maintenance of continued delta-aminolaevulinate synthase synthesis by preventing replenishment of cellular haem either by inhibiting haem synthesis and/or by promoting continuous removal of newly synthesized haem.     

Highlights in haem biosynthesis

The haem biosynthesis pathway continues to provide surprises, from the first enzyme, 5-aminolaevulinic acid synthase, the mRNA of which contains an iron-responsive element, to the last, ferrochelatase, that contains an iron sulphur cluster. 5-Aminolaevulinate dehydratases from animals are zinc-dependent enzymes while those from plants require magnesium. The first X-ray structure of a haem synthesis enzyme, porphobilinogen deaminase, has not only yielded clues about the mechanism of tetrapyrrole assembly but has also provided insight into the molecular basis of the human disease acute intermittent porphyria. Evidence is growing to suggest that a previously unsuspected alternative haem pathway may exist.     

Effect of endotoxin on tryptophan pyrrolase and delta-aminolaevulinate synthase: evidence for an endogenous regulatory haem fraction in rat liver.

Endotoxin was administered to rats at a dose shown previously to stimulate hepatic haem oxygenase activity and to block induction of delta-aminolaevulinate synthase, apparently by causing redistribution of haem from cytochrome P-450 to a regulatory haem pool in the liver. Within 5h of the administration of endotoxin (at a time when the effect of the compound on cytochrome P-450 is maximal) the relative saturation of tryptophan pyrrolase with intrinsic haem rose, from a basal value of 50% to 90%, indicating that 'free' haem had become available. Concurrently, the activity of delta-aminolaevulinate synthase was decreased to 25% of its basal value. Haem oxygenase reached peak activity 13h after endotoxin administration. These findings provide new evidence for the existence of an 'unassigned' hepatic haem fraction, which exchanges with cytochrome P-450 haem and regulates these three enzyme functions.     

Multiple roles of haem in cystathionine β-synthase activity: implications for hemin and other therapies of acute hepatic porphyria

The role of haem in the activity of cystathionine β-synthase (CBS) is reviewed and a hypothesis postulating multiple effects of haem on enzyme activity under conditions of haem excess or deficiency is proposed, with implications for some therapies of acute hepatic porphyrias. CBS utilises both haem and pyridoxal 5′-phosphate (PLP) as cofactors. Although haem does not participate directly in the catalytic process, it is vital for PLP binding to the enzyme and potentially also for CBS stability. Haem deficiency can therefore undermine CBS activity by impairing PLP binding and facilitating CBS degradation. Excess haem can also impair CBS activity by inhibiting it via CO resulting from haem induction of haem oxygenase 1 (HO 1), and by induction of a functional vitamin B₆ deficiency following activation of hepatic tryptophan 2,3-dioxygenase (TDO) and subsequent utilisation of PLP by enhanced kynurenine aminotransferase (KAT) and kynureninase (Kynase) activities. CBS inhibition results in accumulation of the cardiovascular risk factor homocysteine (Hcy) and evidence is emerging for plasma Hcy elevation in patients with acute hepatic porphyrias. Decreased CBS activity may also induce a proinflammatory state, inhibit expression of haem oxygenase and activate the extrahepatic kynurenine pathway (KP) thereby further contributing to the Hcy elevation. The hypothesis predicts likely changes in CBS activity and plasma Hcy levels in untreated hepatic porphyria patients and in those receiving hemin or certain gene-based therapies. In the present review, these aspects are discussed, means of testing the hypothesis in preclinical experimental settings and porphyric patients are suggested and potential nutritional and other therapies are proposed.     

Effect of hexachlorobenzene on haem synthesis

Several drugs are known to induce the liver microsomal mixed-function oxidase system when administered in vivo or even in vitro in cell culture. A sequence of events has been suggested in which the drug is visualized to induce delta-aminolaevulinate synthetase, the first and rate-limiting enzyme of the haem-biosynthetic pathway, which is followed by enhanced haem synthesis and cytochrome P-450 content, facilitating the increase in the drug-metabolizing activity of the liver microsomal fraction. The present studies show that the fungicide hexachlorobenzene, when administered to female rats, can lead to enhanced amounts and rate of synthesis of cytochrome P-450 under conditions when the rate of total haem synthesis has not appreciably altered. The subsequent increase in the rate of total haem synthesis as well as the initial increase in amounts of cytochrome P-450 are brought about under conditions when delta-aminolaevulinate synthetase activity remains constant. However, manifestation of porphyria due to prolonged drug administration is accompanied by a twofold increase in delta-aminolaevulinate synthetase activity. The increase in enzyme activity appears to be due to a decreased degradation rate of the enzyme.     

Defective utilization of haem in selenium-deficient rat liver.

We have previously suggested that an inherent defect in hepatic haem utilization was responsible for the rapid stimulation of hepatic microsomal haem oxygenase activity observed in selenium-deficient rats given phenobarbital, a well known inducer of haem formation. To test this hypothesis, hepatic haem content was deliberately raised in selenium-deficient rats by administration of either tryptophan or allylisopropylacetamide, or by injecting haem itself. We now report that selenium-deficient rats are apparently relatively less efficient in utilizing hepatic haem than normal controls. The findings detailed in the present paper thus indicate that stimulation of hepatic microsomal haem oxygenase activity is indeed a manifestation of abnormal haem utilization in selenium deficiency. This suggests a novel role for selenium in hepatic haem metabolism.     

Conversion of 5-aminolaevulinate into haem by homogenates of human liver. Comparison with rat and chick-embryo liver homogenates.

To assess whether the synthesis of haem can be studied in small amounts of human liver, we measured kinetics of the conversion of 5-aminolaevulinate into haem and haem precursors in homogenates of human livers. We used methods previously developed in our laboratory for studies of rat and chick-embryo livers [Healey, Bonkowsky, Sinclair & Sinclair (1981) Biochem. J. 198, 595-604]. The maximal rate at which homogenates of human livers converted 5-aminolaevulinate into protoporphyrin was only 26% of that for rat, and 58% of that for chick embryo. In the absence of added Fe2+, homogenates of fresh human liver resembled those of chick embryos in that protoporphyrin and haem accumulated in similar amounts, whereas fresh rat liver homogenate accumulated about twice as much haem as protoporphyrin. However, when Fe2+ (0.25 mM) was added to human liver homogenates, mainly haem accumulated, indicating that the supply of reduced iron limited the activity of haem synthase, the final enzyme in the haem-biosynthesis pathway. Addition of the potent iron chelator desferrioxamine after 30 min of incubation with 5-amino[14C]laevulinate stopped further haem synthesis without affecting synthesis of protoporphyrin. Thus the prelabelled haem was stable after addition of desferrioxamine. Since the conversion of 5-amino[14C]laevulinate into haem and protoporphyrin was carried out at pH 7.4, whereas the pH optimum for rat or bovine hepatic 5-aminolaevulinate dehydratase is about 6.3, we determined kinetic parameters of the human hepatic dehydrase at both pH values. The Vmax was the same at both pH values, whereas the Km was slightly higher at the lower pH. Our results indicate that the synthesis of porphyrins and haem from 5-aminolaevulinate can be studied with the small amounts of human liver obtainable by percutaneous needle biopsy. We discuss the implications of our results in relation to use of rat or chick-embryo livers as experimental models for the biochemical features of human acute porphyria.     

Crystal structure of protoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of haem and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX. The membrane-embedded flavoprotein is the target of a large class of herbicides. In humans, a defect in PPO is responsible for the dominantly inherited disease variegate porphyria. Here we present the crystal structure of mitochondrial PPO from tobacco complexed with a phenyl-pyrazol inhibitor. PPO forms a loosely associated dimer and folds into an FAD-binding domain of the p-hydroxybenzoate-hydrolase fold and a substrate-binding domain that enclose a narrow active site cavity beneath the FAD and an alpha-helical membrane-binding domain. The active site architecture suggests a specific substrate-binding mode compatible with the unusual six-electron oxidation. The membrane-binding domains can be docked onto the dimeric structure of human ferrochelatase, the next enzyme in haem biosynthesis, embedded in the opposite side of the membrane. This modelled transmembrane complex provides a structural explanation for the uncoupling of haem biosynthesis observed in variegate porphyria patients and in plants after inhibiting PPO.