What is it about?
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 (Km, Vmax, CLE) 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. The heteromeric hPAH proteins (I65T, R68S, R158Q, I174T, R261Q, V338M, R408W and Y414C) all showed significantly decreased Vmax and CLE values when compared to the homomeric wild-type hPAH enzyme. For both substrates, all calculated Km 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. 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.
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Why is it important?
We report for the first time the kinetic parameters (Km, Vmax, CLE) 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.
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This page is a summary of: Recombinant heteromeric phenylalanine monooxygenase and the oxygenation of carbon and sulfur substrates, Journal of Pharmacy and Pharmacology, March 2011, Wiley, DOI: 10.1111/j.2042-7158.2010.01242.x.
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