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.

Characterization and in vitro evaluation of intestinal absorption of liposomes encapsulating zanamivir.

Zanamivir is currently used for the treatment of H1N1 and H5N1 influenza viruses. Due to its highly hydrophilic property, zanamivir has poor oral bioavailability. Liposomal formulations are known to improve oral absorption of hydrophilic drugs. The present study investigates the effect of liposomes encapsulating zanamivir on the permeation of zanamivir across Caco-2 monolayers. Among the formulations studied, neutral liposomes composed of Phospholipon(®) 90 G and cholesterol at molar ratio of 7:3 gave the highest entrapment efficiency of zanamivir. The extrusion of liposomes loading zanamivir (LZV) resulted in the reduced-size liposomal zanamivir (RLZV), which had mean diameter at 283±42 nm and gave higher encapsulation efficiency of zanamivir at 34.69±6.37% compared to 28.32±5.25%. Transport studies across Caco-2 cell monolayers showed that the apparent permeation coefficients (P(app)) of LZV and RLZV were respectively 2.2- and 3.0-fold greater than that of zanamivir solution. The P(app) of RLZV was 1.4-fold higher than that of LZV. On the basis of these results, liposomes are able to improve permeability of zanamivir across the Caco-2 monolayers, thereby possibly enhancing oral bioavailability of zanamivir.

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.

Phenylalanine 4-monooxygenase and the S-oxidation of S-carboxymethyl-L-cysteine by human cytosolic fractions.

The purpose of this investigation was to reaction phenotype the identity of the cytosolic enzyme responsible for the S-oxidation of S-carboxymethyl-L-cysteine (SCMC) in female human hepatic cytosolic fractions. The identity of this enzyme in the female Wistar rat hepatic cytosolic fraction was found to be phenylalanine 4-monooxygenase (PAH). In pooled female human hepatic cytosolic fractions the calculated K(m) and V(max) for substrate (SCMC) activated PAH was 16.22 +/- 11.31 mM and 0.87 +/- 0.41 nmoles x min(-1) mg(-1). The experimental data modelled to the Michaelis-Menten equation with noncompetitive substrate inhibition. When the cytosolic fractions were activated with lysophophatidylcholine the V(max) increased to 52.31 +/- 11.72 nmoles x min(-1) mg(-1) but the K(m) remained unchanged at 16.53 +/- 2.32 mM. A linear correlation was seen in the production of Tyr and SCMC R/S S-oxide in 20 individual female hepatic cytosolic fractions for both substrate and lysophosphatidylcholine activated PAH (r(s) > 0.96). Inhibitor studies found that the specific chemical and antibody inhibitors of PAH reduced the production of Tyr and SCMC R/S S-oxide in these in vitro PAH assays. An investigation of the mechanism of interaction of SCMC with PAH indicated that the drug was a competitive inhibitor of the aromatic C-oxidation of Phe with a calculated K(i) of 17.23 +/- 4.15 mM. The requirement of BH4 as cofactor and the lack of effect of the specific tyrosine hydroxylase, tryptophan hydroxylase and nitric oxide synthase inhibitors on the S-oxidation of SCMC all indicate that PAH was the enzyme responsible for this biotransformation reaction in human hepatic cytosolic fractions.

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.