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.