PHENYLALANINE 4-MONOOXYGENASE AND THE S-OXIDATION OF S-CARBOXYMETHYL-L-CYSTEINE IN HepG2 CELLS

Abstract

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.

Non-Classical Drug Metabolism Enzymes: The Curious Case of Phenylalanine 4-Monooxygenase

“…A series of detailed studies into the subcellular location, cofactor, activator and inhibitor specificities of "S-carboxymethyl-L-cysteine S-oxygenase", cysteine dioxygenase and phenylalanine 4-monooxygenase in rat liver and HepG2 cells have uncovered substantial differences between "S-carboxymethyl-L-cysteine S-oxygenase and cysteine dioxygenase functioning. However, virtually identical profiles were observed between "S-carboxymethyl-L-cysteine S-oxygenase" and phenylalanine 4-monooxygenase, strongly suggesting that phenylalanine 4-monooxygenase was the enzyme responsible for the Soxidation of S-carboxymethyl-L-cysteine [32,33,34]. These findings are summarised in tabular form ( Table 3).…”

“…A recent investigation of rat phenylalanine 4-monooxygenase (utilising L-phenylalanine and S-carboxymethyl-L-cysteine as substrates) and cysteine dioxygenase (utilising L-cysteine and S-carboxymethyl-L-cysteine as substrates) activities found a negative correlation between the S-oxidation of Lcysteine and S-carboxymethyl-L-cysteine (r s = -0.59, P> 0.05) but a positive linear correlation between the Coxidation of L-phenylalanine and the S-oxidation of Scarboxymethyl-L-cysteine (r s = 0.84, P< 0.001) (Fig. 2) [32,33,34]. When the above data was ranked, allowing a direct relationship between the three enzyme activities to be assessed, the positive linear correlation between "Scarboxymethyl-L-cysteine S-oxygenase" and phenylalanine 4-monooxygenase was retained (r s = 0.95, P<0.001) whereas that between "S-carboxymethyl-L-cysteine S-oxygenase" and cysteine dioxygenase (r s = -0.62) was virtually identical to that seen between cysteine dioxygenase and phenylalanine 4-monooxygenase (r s = -0.70), the latter being two enzymes known to be completely separate gene products with independent and unrelated function (Fig.…”

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

“…The initial suggestion based on the structural similarities between cysteine and S-carboxymethyll-cysteine was that the enzyme, cysteine dioxygenase, was responsible for the reaction. A series of detailed studies on the subcellular location, cofactor, activator, inhibitor and substrate specificities of 'S-carboxymethyl-l-cysteine S-oxygenase', in rat and human hepatic cytosolic fractions, HepG2 cell cytosolic fractions and cDNA expressed human and mouse PAH have implicated this endobiotic metabolism enzyme as the 'S-carboxymethyl-l-cysteine S-oxygenase' [8,9,[18][19][20][21][22]. A recent investigation of rat PAH (utilising l-phenylalanine and S-carboxymethyl-l-cysteine as substrates) and cysteine dioxygenase (utilising l-cysteine and S-carboxymethyl-l-cysteine as substrates) found a negative correlation between the sulfoxidation of l-cysteine and S-carboxymethyl-l-cysteine but a positive linear correlation between the C-oxidation of l-phenylalanine and the sulfoxidation of S-carboxymethyll-cysteine (r s = 0.84, Spearman's Rank correlation coefficient, p < 0.001) [18].…”

“…Until now, there are no published experimental data available to support this suggestion and its origin may be traced to a literature reference containing only an unsubstantiated statement [28]. The role of PAH in the S-oxidation of S-carboxymethyll-cysteine in humans, rat and mouse has now been firmly established [8][9][10][18][19][20][21][22] with regard to the parent compound and three of its known thioether metabolites (S-methyl-l-cysteine, N-acetyl-S-carboxymethyl-l-cysteine and N-acetyl-S-methyll-cysteine). Of the two other known thioether metabolites, thiodiglycolic acid is not a substrate for PAH and the ester glucuronide of S-carboxymethyl-l-cysteine has not been investigated due to its lack of availability [15,29].…”

“…Nor-leucine also undergoes aliphatic C-oxidation to its ε-hydroxy derivative [6,[8][9][10]. The physiological regulation of PAH with regards to l-phenylalanine metabolism occurs through mechanisms 1 and 2.…”