Influence of Steric Bulk and Electrostatics on the Hydroxylation Regiospecificity of Tryptophan Hydroxylase:  Characterization of Methyltryptophans and Azatryptophans as Substrates

Abstract

Tryptophan hydroxylase is a pterin-dependent amino acid hydroxylase that catalyzes the incorporation of one atom of molecular oxygen into tryptophan to form 5-hydroxytryptophan. The substrate specificity and hydroxylation regiospecificity of tryptophan hydroxylase have been investigated using tryptophan analogues that have methyl substituents or nitrogens incorporated into the indole ring. The products of the reactions show that the regiospecificity of tryptophan hydroxylase is stringent. Hydroxylation does not occur at the 4 or 6 carbon in response to changes in substrate topology or atomic charge. 5-Hydroxymethyltryptophan and 5-hydroxy-4-methyltryptophan are the products from 5-methyltryptophan. These products establish that the hydroxylating intermediate is sufficiently potent to hydroxylate benzylic carbons and that the direction of the NIH shift in tryptophan hydroxylase is from carbon 5 to carbon 4. The effects on the V/K values for the amino acids indicate that the enzyme is most sensitive to changes at position 5 of the indole ring. The V(max) values for amino acid hydroxylation differ at most by a factor of 3 from that observed for tryptophan, while the efficiencies of hydroxylation with respect to tetrahydropterin consumption vary 6-fold, consistent with oxygen transfer to the amino acid being partially or fully rate limiting in productive catalysis.

Substrate Hydroxylation by the Oxido–Iron Intermediate in Aromatic Amino Acid Hydroxylases: A DFT Mechanistic Study

“…1 kcal mol -1 lower than the overall effective energy barrier for the formation of the Fe IV =O species (13.9 kcal mol -1 ). These calculated barrier heights are in excellent agreement with the fact that, in most cases, formation of the hydroxylating intermediate appears to represent the rate-limiting step, although there are also examples where oxygen insertion into the amino acid substrate takes on a partial or complete rate-determining role, [3,27] indicating that the two barriers are intrinsically of comparable magnitudes and that their relative order may be modified by the exact choice of enzyme, substrate, and cofactor.…”

“…[25] It has subsequently also been reported that the second main step, the hydroxylation of the aromatic substrate (see below), more specifically the incorporation of the oxygen into the substrate, could, completely or partially, be rate-limiting. [26,27] In the second main reaction step, the aromatic amino acid substrate is hydroxylated by Fe IV =O through an attack by the carbon atom in the aromatic ring on the oxygen atom in Fe IV =O, followed by a subsequent 1,2-hydride transfer, a so-called NIH shift, to form a dienone. [28][29][30] Organic catalysts are not able to perform NIH shifts, and even the low-yield nonspecific hydroxylation of l-Phe by BH 4 in the absence of enzyme occurs without an NIH shift.…”

Mechanism of Aromatic Amino Acid Hydroxylation

“…Other enzymes for which such an intermediate has been invoked (e.g., iso-penicillin synthase and the α-ketoglutarate dependent hydroxylases) are capable of aliphatic hydroxylation (77). Consistent with such an expectation, all three hydroxylases have been shown to catalyze benzylic hydroxylation of methylated aromatic amino acids (54,85,88). In addition, PheH has been reported to catalyze aliphatic hydroxylation (21,46) and epoxidation (23).…”

“…In contrast to the situation with PheH and TyrH, the rate-limiting step in TrpH turnover is oxygen addition to the aromatic ring of the substrate (54). In the hydroxylation of tryptophan, the NIH shift is from C5 to C4 only (58).…”

Mechanism of Aromatic Hydroxylation by an Activated FeIVO Core in Tetrahydrobiopterin‐Dependent Hydroxylases

“…For example, experiments suggest that the nitrogen of the indole could play an important role for the appropriate positioning of the amino acid through an essential hydrogen bonding. [46] Experiments indicate an inverse secondary kinetic isotope effect of 0.93 with 5-2 H-tryptophan, but no inverse isotope effect with 4-2 H-tryptophan (the measured isotope effect was in this case 1.03). [11] It was thus suggested that the isotope effect could be ascribed to the CÀO bond formation during the substrate hydroxylation, which in turn has to involve a higher barrier than the one of the cofactor hydroxylation.…”