Phenylalanine 4-monooxygenase: the "sulfoxidation polymorphism".

1. Consistent differences in the proportion of an orally administered dose of S-carboxymethyl-l-cysteine subsequently excreted in the urine as S-oxide metabolites were reported 40 years ago. This observation suggested the existence of inter-individual variation in the ability to undertake the enzymatic S-oxygenation of this compound. Pedigree studies and investigations employing twin pairs indicated a genetically controlled phenomenon overlaid with environmental influences. It was reproducible and not related to gender or age.2. Studies undertaken in several healthy volunteer cohorts always provided similar results that were not significantly different when statistically analysed. However, when compared to these healthy populations, a preponderance of subjects exhibiting the characteristic of poor sulfoxidation of S-carboxymethyl-l-cysteine was found within groups of patients suffering from various disease conditions. The most striking of these associations were witnessed amongst subjects diagnosed with neurodegenerative disorders; although, underlying mechanisms were unknown.3. Exhaustive investigation has identified the enzyme responsible for this S-oxygenation reaction as the tetrahydrobiopterin-dependent aromatic amino acid hydroxylase, phenylalanine 4-monooxygenase classically assigned the sole function of converting phenylalanine to tyrosine. The underlying principle is discussed that enzymes traditionally associated solely with intermediary metabolism may have as yet unrecognised alternative roles in protecting the organism from potential toxic assault.

The S-oxidation of S-carboxymethyl-L-cysteine in hepatic cytosolic fractions from BTBR and phenylketonuria enu1 and enu2 mice.

Mice that were heterozygous dominant for the enu1 and enu2 mutation in phenylalanine monooxygenase/phenylalanine hydroxylase (PAH) resulted in hepatic PAH assays for S-carboxymethyl-L-cysteine (SCMC) that had significantly increased calculated Km (wild type (wt)/enu1, 1.84-2.12 fold increase and wt/enu2 a 2.75 fold increase in PAH assays). The heterozygous dominant phenotypes showed a significantly reduced catalytic turnover of SCMC (wt/enu1, 6.11 fold decrease and wt/enu2 an 11.25 fold decrease in calculated Vmax). Finally, these phenotypes also had a significantly reduced clearance, CLE (wt/enu1, 13.02 fold and wt/enu2, a 30.80-30.94 fold decrease) The homozygous recessive phenotype (enu1/enu1) was also found to have significantly increased calculated Km (2.16 fold increase), a significantly reduced calculated Vmax (11.35-12.33 fold decrease) and CLE (24.75-25.00 fold decrease). The enu2/enu2, homozygous recessive phenotype had no detectable PAH activity using SCMC as substrate. The identity of the enzyme responsible for the C-oxidation of L-phenylalanine (L-Phe) and the S-oxidation of SCMC in wt/wt (BTBR) mice was identified using monoclonal antibody and selective chemical inhibitors and was found to be PAH. This in vitro mouse hepatic cytosolic fraction metabolism investigation provides further evidence to support the hypothesis that an individual possessing one variant allele for PAH will result in a poor metaboliser phenotype that is unable to produce significant amounts of S-oxide metabolites of SCMC.

Phenylalanine monooxygenase and the sulfur oxygenation of S-carboxymethyl-L-cysteine in mice.

1. The extent of sulfoxidation of the drug, S-carboxymethyl-L-cysteine, has been shown to vary between individuals, with this phenomenon being mooted as a biomarker for certain disease states and susceptibilities. Studies in vitro have indicated that the enzyme responsible for this reaction was phenylalanine monooxygenase but to date no in vivo evidence exists to support this assumption. Using the mouse models of mild hyperphenylalaninamia (enu1 PAH variant) and classical phenylketonuria (enu2 PAH variant), the sulfur oxygenation of S-carboxymethyl-L-cysteine has been investigated. 2. Compared to the wild type (wt/wt) mice, both the heterozygous dominant (wt/enu1 and wt/enu2) mice and the homozygous recessive (enu1/enu1 and enu2/enu2) mice were shown to have significantly increased Cmax, AUC(0-180 min) and AUC(0-∞ min) values (15 - to 20-fold higher). These results were primarily attributable to the significantly reduced clearance of S-carboxymethyl-L-cysteine (13 - to 22-fold lower). 3. Only the wild type mice produced measurable quantities of the parent S-oxide metabolites. Those mice possessing one or more allelic variant showed no evidence of blood SCMC (R/S) S-oxides. These observations support the proposition that differences in phenylalanine hydroxylase activity underlie the variation in S-carboxymethyl-L-cysteine sulfoxidation and that no other enzyme is able to undertake this reaction.

S-carboxymethyl-L-cysteine and it (R/S)-S-oxides in beagle dog plasma and hepatic cytosol.

1. Incubation of beagle hepatic cytosol, under conditions promoting phenylalanine hydroxylase activity, led to the formation of the sulfoxide derivatives of S-carboxymethyl-L-cysteine, N-acetyl-S-carboxymethyl-L-cysteine, S-methyl-L-cysteine and N-acetyl-S-methyl-L-cysteine. Thiodiglycolic acid was not a substrate. Enzyme kinetic parameters (Km, Vmax) were derived indicating S-carboxymethyl-L-cysteine had the greatest clearance; no enantioselective preference was observed for this S-oxygenation reaction. 2. Following oral administration of S-carboxymethyl-L-cysteine to beagle dogs, the parent substance and its sulfoxide were the only compounds identified in the plasma. Pharmacokinetic data have been obtained indicating that the small amount of sulfoxide formed persisted within the body for longer than the parent material, but that the majority of the ingested dose remained in the administered sulfide form. 3. The sulfide moiety within the muco-regulatory drug, S-carboxymethyl-L-cysteine, is thought to be vital as it acts as a free radical scavenger, resulting in the inactive sulfoxide. Additional extensive enyzme-mediated sulfoxidation would decrease the amount of active sulfide available. In the dog this appears to not be an issue, signalling possible exploitation for therapeutic benefit in treating airway disease.

Formation of S-(carboxymethyl)-cysteine in rat liver mitochondrial proteins: effects of caloric and methionine restriction.

Maillard reaction contributes to the chemical modification and cross-linking of proteins. This process plays a significant role in the aging process and determination of animal longevity. Oxidative conditions promote the Maillard reaction. Mitochondria are the primary site of oxidants due to the reactive molecular species production. Mitochondrial proteome cysteine residues are targets of oxidative attack due to their specific chemistry and localization. Their chemical, non-enzymatic modification leads to dysfunctional proteins, which entail cellular senescence and organismal aging. Previous studies have consistently shown that caloric and methionine restrictions, nutritional interventions that increase longevity, decrease the rate of mitochondrial oxidant production and the physiological steady-state levels of markers of oxidative damage to macromolecules. In this scenario, we have detected S-(carboxymethyl)-cysteine (CMC) as a new irreversible chemical modification in mitochondrial proteins. CMC content in mitochondrial proteins significantly correlated with that of the lysine-derived analog N (ε)-(carboxymethyl)-lysine. The concentration of CMC is, however, one order of magnitude lower compared with CML likely due in part to the lower content of cysteine with respect to lysine of the mitochondrial proteome. CMC concentrations decreases in liver mitochondrial proteins of rats subjected to 8.5 and 25 % caloric restriction, as well as in 40 and 80 % methionine restriction. This is associated with a concomitant and significant increase in the protein content of sulfhydryl groups. Data presented here evidence that CMC, a marker of Cys-AGE formation, could be candidate as a biomarker of mitochondrial damage during aging.

Post-translational activation of human phenylalanine 4-monooxygenase from an endobiotic to a xenobiotic enzyme by reactive oxygen and reactive nitrogen species.

An investigation into the post-translational activation of cDNA-expressed human phenylalanine 4-monooxygenase and human hepatic cytosolic fraction phenylalanine 4-monooxygenase activity with respect to both endobiotic metabolism and xenobiotic metabolism revealed that the reactive oxygen species (hydrogen peroxide and hydroxyl radical) and reactive nitrogen species (nitric oxide and peroxynitrite) could elicit the post-translational activation of the enzyme with respect to both of these biotransformation reactions. In virtually all instances, the K(m) values were decreased and the V(max) values were increased; the only exceptions observed being with hydrogen peroxide and L-phenylalanine. These effects were shown to occur at activator concentrations known to exist in physiological situations and, hence, suggest that reactive oxygen and reactive nitrogen species may cause, and may be involved with, the post-translational activation of phenylalanine 4-monooxygenase within the human body. This mechanism, in response to free-radical bursts, may enable the enzyme to expand its substrate range and to process certain xenobiotics as and when required.

The activity of wild type and mutant phenylalanine hydroxylase with respect to the C-oxidation of phenylalanine and the S-oxidation of S-carboxymethyl-L-cysteine.

The involvement of the enzyme, phenylalanine hydroxylase (PAH), in the S-oxidation of S-carboxymethyl-L-cysteine (SCMC) is now firmly established in man and rat. However, the underlying role of the molecular genetics of PAH in dictating and influencing the S-oxidation polymorphism of SCMC metabolism is as yet unknown. In this work we report that the S-oxidation of SCMC was dramatically reduced in the tetrahydrobiopterin (BH(4)) responsive mutant PAH proteins (I65T, R68S, R261Q, V388M and Y414C) with these enzymes possessing between 1.2% and 2.0% of the wild type PAH activity when SCMC was used as substrate. These same mutant proteins express between 23% and 76% of the wild type PAH activity when phenylalanine was used as the substrate. The PAH mutant proteins (R158Q, I174T and R408W) that result in the classical phenylketonuria (PKU) phenotype expressing 0.2-1.8% of the wild type PAH activity when using phenylalanine as substrate were found to have <0.1% of the wild type PAH activity when SCMC was used as the substrate. Mutations that result in PAH proteins retaining some residual PAH activity with phenylalanine as substrate have <2.0% residual activity when SCMC was used as a substrate. This investigation has led to the hypothesis that the S-oxidation polymorphism in man is a consequence of an individual carrying one mutant PAH allele which has resulted in the loss of the ability of the residual PAH protein to undertake the S-oxidation of SCMC in vivo.

Human phenylalanine monooxygenase and thioether metabolism.

OBJECTIVES: The substrate specificity of wild-type human phenylalanine monooxygenase (wt-hPAH) has been investigated with respect to the mucoactive drug, S-carboxymethyl-L-cysteine and its thioether metabolites. The ability of wt-hPAH to metabolise other S-substituted cysteines was also examined. METHODS: Direct assays of PAH activity were by HPLC with fluorescence detection; indirect assays involved following disappearance of the cofactor by UV spectroscopy. KEY FINDINGS: wt-hPAH catalysed the S-oxygenation of S-carboxymethyl-L-cysteine, its decarboxylated metabolite, S-methyl-L-cysteine, and both their corresponding N-acetylated forms. However, thiodiglycolic acid was not a substrate. The enzyme profiles for both phenylalanine and S-carboxymethyl-L-cysteine showed allosteric kinetics at low substrate concentrations, with Hill constants of 2.0 and 1.9, respectively, for the substrate-activated wt-hPAH. At higher concentrations, both compounds followed Michaelis-Menten kinetics, with non-competitive substrate inhibition profiles. The thioether compounds, S-ethyl-L-cysteine, S-propyl-L-cysteine and S-butyl-L-cysteine were all found to be substrates for phenylalanine monooxygenase. CONCLUSIONS: Phenylalanine monooxygenase may play a wider role outside intermediary metabolism in the biotransformation of dietary-derived substituted cysteines and other exogenous thioether compounds.

Comparison of the sulfur-oxygenation of cysteine and S-carboxymethyl-l-cysteine in human hepatic cytosol and the rôle of cysteine dioxygenase.

OBJECTIVES: To determine the Km , Vmax , cofactor, activator and inhibitor requirements of human cysteine dioxygenase and S-carboxymethyl-l-cysteine S-oxygenase with respect to both l-Cysteine and S-carboxymethyl-l-cysteine as substrates. METHODS: In vitro human hepatic cytosolic fraction enzyme assays were optimised for cysteine dioxygenase activity using l-Cysteine as substrate and the effect of various cofactors, activators and inhibitors on the S-oxidations of both l-Cysteine and S-carboxymethyl-l-cysteine were investigated. KEY FINDINGS: The results of the in vitro reaction phenotyping investigation found that although both cysteine dioxygenase and S-carboxymethyl-l-cysteine S-oxygenase required Fe2+ for catalytic activity both enzymes showed considerable divergence in cofactor, activator and inhibitor specificities. Cysteine dioxygenase has no cofactor but uses NAD+ and NADH(H+ ) as pharmacological chaperones and is not inhibited by S-carboxymethyl-l-cysteine. S-carboxymethyl-l-cysteine S-oxygenase requires tetrahydrobiopterin as a cofactor, is not activated by NAD+ and NADH(H+ ) but is activated by l-Cysteine. Additionally, the sulfydryl alkylating agent, N-ethylmaleimide, activated carboxymethyl-l-cysteine S-oxygenase but inhibited cysteine dioxygenase. CONCLUSIONS: Human hepatic cytosolic fraction cysteine dioxygenase activity is not responsible for the S-oxidation of the substituted cysteine, S-carboxymethyl-l-cysteine.

Phenylalanine 4-monooxygenase and the S-oxidation of S-carboxymethyl-L-cysteine in HepG2 cells.

The role of phenylalanine 4-monooxygenase (PAH) in the S-oxidation of S-carboxymethyl-L-cysteine (SCMC) in the rat has now been well established in rat cytosolic fractions in vitro. However, the role of PAH in the S-oxidation of SCMC in human cytosolic fractions or hepatocytes has yet to be investigated. The aim of this investigation was to analyse the kinetic parameters of PAH oxidation of both L-phenylalanine (Phe) and SCMC in the human HepG2 cell line in order to investigate the use of these cells as a model for the cellular regulation of SCMC S-oxidation. The experimentally determined Km and V(max) were 7.14 +/- 0.32 mM and 0.85 +/- 0.32 nmole Tyr formed min(-1) x mg protein(-1) using Phe as substrate. For SCMC the values were 25.24 +/- 5.91 mM and 0.79 +/- 0.09 nmole SCMC (RIS) S-oxides formed min(-1) x mg protein(-1). The experimentally determined Km and V(max) for the cofactor BH4 were 6.81 +/- 0.21 microM and 0.41 +/- 0.004 nmole Tyr formed min(-1) x mg protein(-1) for Phe and 7.24 +/- 0.19 microM and 0.42 +/- 0.002 nmole SCMC (R/S) S-oxides formed min(-1) x mg protein(-1) for SCMC. The use of various PAH inhibitors confirmed that HepG2 cells contained PAH and that the enzyme was capable of converting SCMC to its (R) and (S) S-oxide metabolites in an in vitro PAH assay. Thus HepG2 cells have become a useful additional tool for the investigation of the cellular regulation of PAH in the S-oxidation of SCMC.

Re-evaluation of the metabolism of carbocisteine in a British white population.

It has been claimed that the amino acid derivative carbocisteine is predominantly metabolized by sulfoxidation and that this pathway exhibits a genetic polymorphism. Moreover, those subjects with a 'poor metabolizer' phenotype have been thought to have a genetic predisposition to developing certain diseases. We have confirmed the observations of others that this marker drug does not undergo significant S-oxidation. Furthermore, a novel urinary metabolite, S-(carboxymethylthio)-L-cysteine (CMTC) has recently been identified. To determine if a genetic polymorphism for this biotransformation pathway exists, metabolic ratios (% urinary excretion carbocisteine/% urinary excretion CMTC) for 120 healthy volunteers were assessed using high-performance thin-layer chromatography. Urinary excretion of the parent drug ranged from 6% of the dose administered to 56% (mean +/- SD, 23.4 +/- 0.8%). No cysteinyl sulfoxide metabolites were identified in the urine samples. The amount excreted as CMTC exhibited a 12-fold variation but only accounted for mean of 4.4% (1-12%) of the dose given. Two individuals initially had high metabolic ratios (> 30), however, on rechallenge both their MRs were less than 5. Therefore, carbocisteine is not an appropriate probe drug for sulfoxidation. The formation of the novel metabolite CMTC appears to exhibit polymorphism, although the considerable intra-subject variation for its formation does not allow assignment of a phenotype.

Unusual pKa of the carboxylate at the putative catalytic position of the thermophilic F1-ATPase beta subunit determined by 13C-NMR.

Glutamic acid-190 in the beta subunit of F1-ATPase from thermophilic Bacillus PS-3 (TF1) was reported to be essential for the ATPase activity. The mutant TF1beta subunit in which Glu-190 had been substituted by cysteine was carboxymethylated with 13C-labeled monoiodoacetic acid. The pKa value of the carboxymethylene group at the 190 position was determined as 5.6 +/- 0.4 by 13C-NMR. On the basis of this value, the pKa of the carboxylate of Glu-190 of the TF1beta subunit was estimated to be 6.8 +/- 0.5. The unusually high pKa could play a role in the catalytic mechanism of F1-ATPase.

Xenobiotic metabolism in Alzheimer's disease.

Using 5 methods, we assessed the ability of patients with a clinical diagnosis of Alzheimer's disease (AD) to handle xenobiotics. Patients with AD, compared with controls, have reduced sulfoxidation of the probe drug S-carboxymethyl-L-cysteine; they also form less of the sulfate conjugate of acetaminophen. In addition, they have lower activity of the enzyme thiolmethyltransferase. In contrast, the capacity to oxidize debrisoquin and to acetylate sulfamethazine was normal. These findings suggest that a major risk factor for the development of AD is a skewed capacity for xenobiotic metabolism especially of compounds containing sulfur.

14C-labeling of synthetic peptides.

Two methods are described for the labeling of synthetic peptides using iodo[14C]acetic acid. The first procedure may be employed when the synthetic fragment contains a cysteine with a free sulfhydryl group. Alternatively, a commercial amino-protected cysteine may be carboxymethylated using radioactive iodoacetic acid. This derivative can be added to the growing peptide chain in the manual or automatic solid-phase synthesis of the fragment.

Pharmacokinetic behavior of S-carboxymethylcysteine-Lys in patients with chronic bronchitis.

A mass fragmentographic technique was used to study the pharmacokinetic behavior of SCMC-Lys in patients with acute exacerbations of chronic bronchitis and with dense expectoration. Serum and urine levels, as well as bronchial mucus levels and their correlations, were determined. The data suggest that SCMC-Lys diffuses well into bronchial mucus, a useful feature for a mucolytic drug.

Quantitative investigation of copper(II) and zinc(II) complexes with S-carboxymethyl-L-cysteine and computer-simulated appraisal of their potential significance in vivo.

S-carboxymethyl-L-cysteine (SCC) is a mucolytic agent extensively used in the treatment of respiratory tract disorders. Some of the undesirable side effects observed during SCC therapy being reminiscent of symptoms characteristic of copper and zinc imbalances, the objective of this paper was to test the possible interference of SCC with the metabolism of these two metals. Copper(II)- and zinc(II)-SCC complex equilibria have thus been investigated under physiological conditions by means of classical potentiometry combined with computer-assisted calculation techniques. Formation constants derived from these studies have then been used to simulate 1) the potential influence of SCC on the distribution of the above metals in blood plasma and 2) the extent to which gastrointestinal interactions between the drug and each metal ion in turn are likely to affect the bioavailability of each other. The results of these simulations show that 1) plasma therapeutic levels of SCC are not likely to induce dramatic changes in the distributions of copper(II) and zinc(II) low molecular weight fractions, 2) the gastrointestinal distribution of the drug is not affected by standard dietary doses of these metals, and 3) in contrast, therapeutic concentrations of SCC are capable of mobilizing significant fractions of both metals into tissue-diffusible electrically neutral complexes. In conclusion significant depletions of neither copper nor zinc are to be expected from oral administration of SCC. While the drug may to some extent facilitate the excretion of Cu2+ and Zn2+ ions from blood plasma, its gastrointestinal influence is, on the contrary, favorable to a better absorption of these two metals.

RIT 2214, a new biosynthetic penicillin produced by a mutant of Cephalosporium acremonium.

A number of lysine-requiring auxotrophs of Cephalosporium acremonium were investigated for incorporation of side-chain precursors and for accumulation of beta-lactam compounds. One of the auxotrophs, Acremonium chrysogenum ATCC 20389, producing cephalosporin C and penicillin N only if grown in media supplemented with DL-alpha-amino-adipic acid (DL-alpha-AAA), was found to use L-S-carboxymethylcysteine (L-CMC) as a side-chain precursor for the synthesis of a new penicillin (RIT 2214). No corresponding cephalosporin was detected. The penicillin present in the culture filtrate, was concentrated by adsorption on activated carbon and successive column chromatography on Amberlite IRA-68 and Amberlite XAD-4. Final purification was achieved by cellulose column chromatography. RIT 2214 was identified as 6-(D)-[(2-amino-2-carboxy)-ethylthio]-acetamido]-penicillanic acid by spectral analysis, bioactivity spectrum, elucidation of side-chain structure and finally by semisynthesis. Its biological properties were also evaluated.

Lack of congruence between cysteine dioxygenase activity and S-carboxymethyl-L-cysteine S-oxidation activity in rat cytosol.

The identity of the enzyme(s) responsible for the S-oxidation of the mucoactive drug S-carboxymethyl-L-cysteine (SCMC) is unknown but the protein(s) are a susceptibility factor for a number of chronic degenerative diseases. The structural similarities between the amino acid L-cysteine and SCMC have raised the possibility that cysteine dioxygenase (CDO) may be responsible for this biotransformation reaction. Both CDO and SCMC S-oxygenase were found to require Fe2+ for enzymatic activity, and both enzyme activities were inhibited by Fe2+ and Fe3+ chelators. However, sulphydryl group modification of the enzymes resulted in the activation of the S-oxidation of SCMC but inhibition of the S-oxidation of L-cysteine. When the two enzyme activities were quantified in 20 female hepatic cytosolic fractions no linear correlation in the production of their respective metabolites was seen. The results of this investigation indicate that CDO is not responsible for the S-oxidation of SCMC in the rat.