Phenylalanine hydroxylase from Chromobacterium violaceum (CvPheH) is a non-heme iron monooxygenase that catalyzes the hydroxylation of phenylalanine to tyrosine. In this study, we used deuterium kinetic isotope effects to probe the chemical mechanisms of aromatic and benzylic hydroxylation to compare the reactivities of bacterial and eukaryotic aromatic amino acid hydroxylases. The Dkcat value for the reaction of CvPheH with [ 2H5]phenylalanine is 1.2 with 6-methyltetrahydropterin and 1.4 with 6,7-dimethyltetrahydropterin. With the mutant enzyme I234D, the Dkcat value decreases to 0.9 with the latter pterin; this is likely to be the intrinsic effect for addition of oxygen to the amino acid. The isotope effect on the subsequent tautomerization of a dienone intermediate was determined to be 5.1 by measuring the retention of deuterium in tyrosine produced from partially deuterated phenylalanine; this large isotope effect is responsible for the normal effect on kcat. The isotope effect for hydroxylation of the methyl group of 4-CH3-phenylalanine, obtained from the partitioning of benzylic and aromatic hydroxylation products, is 10. The temperature dependence of this isotope effect establishes the contribution of hydrogen tunneling to benzylic hydroxylation by this enzyme. The results presented here provide evidence that the reactivities of the prokaryotic and eukaryotic hydroxylases are similar and further define the reactivity of the iron center for the family of aromatic amino acid hydroxylases.
|Original language||English (US)|
|Number of pages||7|
|State||Published - Oct 21 2008|
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