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.

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 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.

Disulphide linkage in mouse ST6Gal-I: determination of linkage positions and mutant analysis.

All cloned sialyltransferases from vertebrates are classified into four subfamilies and are characterized as having type II transmembrane topology. The catalytic domain has highly conserved motifs known as sialylmotifs. Besides sialylmotifs, each family has several unique conserved cysteine (Cys) residues mainly in the catalytic domain. The number and loci of conserved amino acids, however, differ with each subfamily, suggesting that the conserved Cys-residues and/or disulphide linkages they make may contribute to linkage specificity. Using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF)-mass spectrometry, the present study performed disulphide linkage analysis on soluble mouse ST6Gal-I, which has six Cys-residues. Results confirmed that there were no free Cys-residues, and all six residues contributed to disulphide linkage formation, C(139)-C(403), C(181)-C(332) and C(350)-C(361). Study of single amino acid-substituted mutants revealed that the disulphide linkage C(181)-C(332) was necessary for molecular expression of the enzyme, and that the disulphide linkage C(350)-C(361) was necessary for enzyme activity. The remaining disulphide linkage C(139)-C(403) was not necessary for enzyme expression or for activity, including substrate specificity. Crystallographic study of pig ST3Gal I has recently been reported. Interestingly, the loci of disulphide linkages in ST6Gal-I differ from those in ST3Gal I, suggesting that the linkage specificity of sialyltransferase may results from significant structural differences, including the loci of disulphide linkages.

Recombinant heteromeric phenylalanine monooxygenase and the oxygenation of carbon and sulfur substrates.

OBJECTIVES: The aim of this investigation was to provide in-vitro enzyme kinetic data to support the hypothesis that the in-vivo heterozygous dominant phenotype for phenylalanine monooxygenase (hPAH) was responsible for the S-oxidation polymorphism in the metabolism of S-carboxymethyl-l-cysteine reported in humans. Using a dual-vector expression strategy for the co-production of wild-type and mutant human hPAH subunits we report for the first time the kinetic parameters (K(m) , V(max) , CL(E) ) for the C-oxidation of l-phenylalanine and the S-oxidation of S-carboxymethyl-l-cysteine in homomeric wild-type, heteromeric mutant and homomeric mutant hPAH proteins in vitro. METHODS: A PRO(TM) dual-vector bacterial expression system was used to produce the required hPAH proteins. Enzyme activity was determined by HPLC with fluorescence detection. KEY FINDINGS: The heteromeric hPAH proteins (I65T, R68S, R158Q, I174T, R261Q, V338M, R408W and Y414C) all showed significantly decreased V(max) and CL(E) values when compared to the homomeric wild-type hPAH enzyme. For both substrates, all calculated K(m) values were significantly higher than homomeric wild-type hPAH enzyme, with the exception of I65T, R68S and Y414C heteromeric hPAH proteins employing l-phenylalanine as substrate. CONCLUSIONS: The net outcome for the heteromeric mutant hPAH proteins was a decrease significantly more dramatic for S-carboxymethyl-l-cysteine S-oxidation (1.0-18.8% of homomeric wild-type hPAH activity) when compared to l-phenylalanine C-oxidation (25.9-52.9% of homomeric wild-type hPAH activity) as a substrate. Heteromeric hPAH enzyme may be related to the variation in S-carboxymethyl-l-cysteine S-oxidation capacity observed in humans.

A comprehensive understanding of thioTEPA metabolism in the mouse using UPLC-ESI-QTOFMS-based metabolomics.

ThioTEPA, an alkylating agent with anti-tumor activity, has been used as an effective anticancer drug since the 1950s. However, a complete understanding of how its alkylating activity relates to clinical efficacy has not been achieved, the total urinary excretion of thioTEPA and its metabolites is not resolved, and the mechanism of formation of the potentially toxic metabolites S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA) remains unclear. In this study, the metabolism of thioTEPA in a mouse model was comprehensively investigated using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based-metabolomics. The nine metabolites identified in mouse urine suggest that thioTEPA underwent ring-opening, N-dechloroethylation, and conjugation reactions in vivo. SCMC and TDGA, two downstream thioTEPA metabolites, were produced from thioTEPA from two novel metabolites 1,2,3-trichloroTEPA (VII) and dechloroethyltrichloroTEPA (VIII). SCMC and TDGA excretion were increased about 4-fold and 2-fold, respectively, in urine following the thioTEPA treatment. The main mouse metabolites of thioTEPA in vivo were TEPA (II), monochloroTEPA (III) and thioTEPA-mercapturate (IV). In addition, five thioTEPA metabolites were detected in serum and all shared similar disposition. Although thioTEPA has a unique chemical structure which is not maintained in the majority of its metabolites, metabolomic analysis of its biotransformation greatly contributed to the investigation of thioTEPA metabolism in vivo, and provides useful information to understand comprehensively the pharmacological activity and potential toxicity of thioTEPA in the clinic.

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.

Phenylalanine 4-monooxygenase and the role of endobiotic metabolism enzymes in xenobiotic biotransformation.

Phenylalanine 4-monooxygenase is the key enzyme in the sulfoxidation of the thioether drug S-carboxymethyl-l-cysteine and its thioether metabolites, S-methyl-l-cysteine, N-acetyl-S-carboxymethyl-l-cysteine and N-acetyl-S-methyl-l-cysteine in humans, and a number of other mammalian species. The kinetics constants of the sulfoxidation reaction (K(m), V(max) and CL(E)) have been investigated in cytosolic fractions derived from rat and human liver, in cytosolic fractions of HepG2 cells and using both human and mouse cDNA expressed phenylalanine 4-monooxygenase. Differences in K(m), V(max) and CL(E) of S-carboxymethyl-l-cysteine have been seen in HepG2 cells and human and mouse cDNA expressed phenylalanine 4-monooxygenase when compared to both rat and human hepatic cytosolic fractions. The association of the genetic polymorphism in the sulfoxidation of S-carboxymethyl-l-cysteine is highlighted with particular reference to this biotransformation reaction as being a biomarker of disease susceptibility in Parkinson's, Alzheimer's and motor neurone diseases and in rheumatoid arthritis. The possible underlying molecular genetics of the sulfoxidation polymorphism is also discussed in relation to the known allelic frequencies of phenylalanine 4-monooxygenase. Finally, the new found role phenylalanine 4-monooxygenase plays in xenobiotic metabolism is discussed.

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.

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.

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.

Phenylalanine hydroxylase: possible involvement in the S-oxidation of S-carboxymethyl-l-cysteine.

Activated phenylalanine 4-monooxygenase, phenylalanine hydroxylase (PAH), is known to be involved in the S-oxidation of a number of sulfide compounds. One of these compounds, S-carboxymethyl-l-cysteine (SCMC), is currently used for the treatment of chronic obstructive pulmonary disease and otitis media with effusion as a mucolytic agent, and the S-oxides are the major metabolites found in urine. However, the enzyme catalyzing the S-oxidation of SCMC has yet to be identified. Here we report on the role of nonactivated phenylalanine 4-monooxygenase activity in rat liver cytosol in the S-oxidation of SCMC. Linearity of the enzyme assays was seen for both time (0-16 min) and cytosolic protein concentration (0.1-0.5mg/ml). The calculated K(m) and V(max) values for the formation of SCMC (S) S-oxide were 3.92+/-0.15 mM and 1.10+/-0.12 nmol SCMC (S) S-oxide formed/mg protein/min, respectively. The calculated K(m) and V(max) values for the formation of SCMC (R) S-oxide were 9.18+/-1.13 mM and 0.46+/-0.11 nmol SCMC (R) S-oxide formed/mg protein/min, respectively. These results indicate that in the female Wistar rat, nonactivated PAH showed a stereospecific preference for the formation of the (S) S-oxide metabolite of SCMC against the (R) S-oxide metabolite of SCMC.

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

The identity of the enzyme responsible for the S-oxidation of the mucolytic S-substituted L-cysteine drug, S-carboxymethyl-L-cysteine (SCMC), has been actively investigated for the last 10 years. A genetic polymorphism exists in the oxidation of the thioether moiety that has been identified as a disease susceptibility factor in a number of degenerative diseases. This polymorphism has also been implicated in the wide variation in clinical response to SCMC therapy in man. To date little is known about the molecular enzymology of this reaction but a previous investigation revealed that rat activated phenylalanine 4-monooxygenase (PAH) could S-oxidise both Met- and S-methyl-L-cysteine (SMC) to their S-oxide metabolites. We have investigated the hypothesis that SCMC was also a substrate for activated PAH in the cytosolic faction of the Wistar rat. 1. Substrate and inhibitor investigation revealed that SCMC was a substrate for activated PAH activity in vitro. 2. The large aromatic amino acid hydroxylase monoclonal antibody and the Fe3+ chelator, deferoxamine, completely inhibited both Phe and SCMC oxidation to their respective metabolites. 3. Analysis of the Dixon plots revealed that both Phe and SCMC competitively inhibited each other's oxidation. 4. Correlation studies showed that the rate of production of Tyr was positively correlated to the production of both SCMC and SMC S-oxides in 20 female Wistar rat hepatic cytosolic fractions. These results strongly support the hypothesis that PAH is the enzyme responsible for SCMC S-oxidation in the rat.