Insights into the Catalytic Mechanisms of Phenylalanine and Tryptophan Hydroxylase from Kinetic Isotope Effects on Aromatic Hydroxylation

Abstract

Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. ]-phenylalanine, are identical for Δ117PheH and Δ117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the D k cat value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6-to7-fold that for hydroxylation. In addition, there is a slight (∼34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With 2 H 5 -indole-tryptophan as a substrate for Δ117PheH, the D k cat value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the D k cat values are within error of those for Δ117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine.Phenylalanine hydroxylase (PheH 1 ), tyrosine hydroxylase (TyrH), and tryptophan hydroxylase (TrpH) form a small family of nonheme iron monooxygenases (1). These enzymes catalyze

Characterization of metal ligand mutants of phenylalanine hydroxylase: Insights into the plasticity of a 2-histidine-1-carboxylate triad

“…For all three enzymes, hydroxylation of the physiological substrate occurs via an electrophilic aromatic substitution reaction with an activated oxygen species [13][14][15]. In the case of TyrH the latter has been shown to be an Fe(IV) species [16], consistent with the proposed involvement of an Fe(IV)O as the hydroxylating intermediate in all three enzymes [12].…”

Iron‐Promoted ortho‐ and/or ipso‐Hydroxylation of Benzoic Acids with H2O2

“…Competition experiments also showed no preference of 1 in reacting with either protio or deuterio isotopomers, so C À H bond breaking on the phenyl ring cannot be a rate limiting step in this transformation. [36] Similarly small H/D KIE values ( 1) were also reported for other aromatic oxidations promoted by biological and synthetic iron centers, [8,27,29,[61][62][63][64] and interpreted in terms of either a rate-limiting formation of an iron-oxo intermediate [29] or a rate-limiting electrophilic attack of the aromatic ring by a metal-based oxidant (a process that is typically characterized by only a small but inverse isotope effect). [27,61,62,65,66] 18 O-labeling experiments with H 2 18 O 2 unambiguously showed the incorporation of one oxygen atom into the phenolate or salicylate product derived from the oxidations of benzoic acids, underscoring the fact that hydrogen peroxide is the sole source of the oxygen atom incorporated into the products.…”

Versatility of biological non-heme Fe(II) centers in oxygen activation reactions

“…Two electrons are required to break the O-O bond to generate the high valent iron oxo species. It is postulated that the intermediate in this case contains a peroxo bridge between the Fe(II) and the tetrahydropterin 74 . Thus, an intermediate iron-superoxo or peroxo species is not formally required, although one is likely to transiently form.…”