A Mechanism for Hydroxylation by Tyrosine Hydroxylase Based on Partitioning of Substituted Phenylalanines

Abstract

The iron-containing enzyme tyrosine hydroxylase catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine. A series of 4-X-substituted (X = H, F, Br, Cl, CH3, or CH3O) phenylalanines have been characterized as substrates to gain insight into the mechanism of hydroxylation. Multiple hydroxylated products were formed in most cases. As the size of the substituent at the 4-position increased, the site of hydroxylation switched from the 4- to the 3-position of the aromatic ring. The total amount of product formed with each amino acid showed a very good correlation with the sigma parameter of the substituent, with rho values of -4.3 +/- 0.7 or -5.6 +/- 0.8 when tetrahydrobiopterin or 6-methyltetrahydropterin, respectively, was used as cosubstrate. These values are consistent with a highly electron deficient transition state for hydroxylation. Oxygen addition at the 4-position resulted in either elimination of the substituent to form tyrosine or an NIH shift to form the respective 3-X-tyrosine. The relative amount of the product due to an NIH shift decreased in the order Br > CH3 > Cl >> F approximately CH3O approximately 0. A chemical mechanism for hydroxylation by tyrosine hydroxylase is presented to account for product formation from the various 4-substituted phenylalanines.

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].…”

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).…”

“…This is consistent with a kinetic model (Scheme 5) in which the rate of formation of the Fe(IV)O intermediate is insensitive to the reactivity of the amino acid substrate, but its subsequent fate is determined by partitioning between productive hydroxylation and unproductive breakdown. When the relative amount of hydroxylated amino acid formed was determined for a series of 4-substituted phenylalanines, there was a good correlation between the relative rate constants for hydroxylation and the σ values of the substituents, with an average ρ value of −5 for tetrahydrobiopterin and 6-methyltetrahydropterin (85). This result was interpreted as evidence for a cationic transition state for oxygen addition to the aromatic ring.…”

Catabolism of Arylboronic Acids by Arthrobacter nicotinovorans Strain PBA

“…In some oxygenase-catalyzed reactions, the position of the oxygen atom in the product can differ from the position of the substituent it replaces via a mechanism known as the NIH shift (12,17,26). Mass spectral analysis of the product would not readily reveal this.…”

“…Oxygenases are widespread in soil microorganisms and are widely known to displace different substituents from alicyclic and aromatic rings to generate hydroxylated intermediates (4, 11, 34; L. B. M. Ellis and L. P. Wackett, The University of Minnesota Biocatalysis/Biodegradation Database, http://umbbd.ahc.umn.edu/, 22 December 2002). For example, aromatic rings containing fluorine, chlorine, bromine, cyano, nitro, amino, sulfonate, and carboxylate substituents are oxygenated to produce hydroxylated products with displacement of the original substituent (12,17,34).…”