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.
Boonyapiwat
Boontarika
B
Pharmaceutical Sciences Research Division, School of Health and Life Sciences, King's College London, London, UK.
Panagopoulos
Panayotis
P
Jones
Hevfin
H
Mitchell
Stephen C
SC
Forbes
Ben
B
Steventon
Glyn B
GB
D015082:2,2'-Dipyridyl / Q000378:metabolism; D024322:Amino Acids, Aromatic / Q000378:metabolism; D002233:Carbocysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D002460:Cell Line; D003067:Coenzymes / Q000378:metabolism; D003544:Cysteic Acid / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D003676:Deferoxamine / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D007502:Iron Chelating Agents / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
D024322:Amino Acids, Aromatic; D003067:Coenzymes; D004791:Enzyme Inhibitors; D007502:Iron Chelating Agents; C018243:S-(2-carboxyethyl)cysteine; D014443:Tyrosine; D010649:Phenylalanine; D015082:2,2'-Dipyridyl; D002233:Carbocysteine; D003544:Cysteic Acid; D008715:Methionine; D010651:Phenylalanine Hydroxylase; D003676:Deferoxamine