Intrinsic Deuterium Isotope Effects on Benzylic Hydroxylation by Tyrosine Hydroxylase

Abstract

Tyrosine hydroxylase (TyrH) is a mononuclear, non-heme iron monooxygenase that catalyzes the pterin-dependent hydroxylation of tyrosine to dihydroxyphenylalanine. When 4-methylphenylalanine is used as a substrate for TyrH, 4-hydroxymethylphenylalanine is one of the amino acid products. To examine the mechanism of benzylic hydroxylation, the products and their isotopic compositions were determined with 4-methylphenylalanines containing a mono-, di-, or trideuterated methyl group as substrates. Intrinsic primary and secondary deuterium isotope effects for benzylic hydroxylation of 9.6 +/- 0.9 and 1.21 +/- 0.08, respectively, were derived from the data. The magnitudes of these isotope effects are consistent with quantum mechanical tunneling of the hydrogen. The similarity of the effects to those seen for benzylic hydroxylation by other enzymes supports a mechanism where a high valence iron-oxo species, Fe(IV)=O, is the hydroxylating intermediate.

Mechanism of Aromatic Amino Acid Hydroxylation

“…Only in the case of benzylic hydroxylation by TyrH has the mechanism been investigated in detail. Analysis of the products when 4-methylphenylalanine containing one, two, or three deuterium atoms in the methyl group was used as a substrate allowed determination of the intrinsic primary and secondary deuterium isotope effects for the hydroxylation step (89). The secondary isotope effect of 1.2 demonstrated that there is significant rehybridization of the methyl carbon in the transition state for hydroxylation; such a result is most consistent with the formation of a benzylic radical intermediate by abstraction of a hydrogen atom from the methyl group by the electrophilic Fe (IV)O species, followed by radical rebound of a hydroxyl radical to form the hydroxylated product (Scheme 6).…”

Multidimensional Tunneling, Recrossing, and the Transmission Coefficient for Enzymatic Reactions

“…39 Siebrand and Smedarchina 149 have suggested that the rate constants and their T dependence reflect the influence of kinetic steps prior to the proton transfer. 150 tyrosine hydroxylase, 290 andyeastalcoholdehydrogenase(YADH). 16,67,74,96 Large KIEs have also been reported for the xylene hydroxylation by cytochrome P-450 291 and dopamine β-monooxygenase.…”

Coordination and Bioinorganic Chemistry of Aryl-Appended Tris(2-Pyridylmethyl)amine Ligands

“…[55][56][57][58][59][60][61][62][63] In the catalytic cycle of these enzymes an Fe(IV)=O moiety has been proposed to act as an electrophile toward the aromatic substrate. [55,63,64] In a model study of functional relevance to these enzymes, it was found that treatment of an acetonitrile solution of 4 (Figure 3(a)) with t BuOOH (3 eq) under an argon atmosphere results in the oxidative hydroxylation of the ligand phenyl ring (Scheme 2). [23,35] To isolate the final product of this reaction, base (NEt 3 ) and NaBPh 4 were added in wet methanol, which resulted in the deposition of [(6-C 6 [35] decays over a period of $ 4 hours at À60 C to give a second intermediate prior to the formation of 37.…”