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 σ parameter of the substituent, with ρ 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 ∼ CH₃O ∼ 0. A chemical mechanism for hydroxylation by tyrosine hydroxylase is presented to account for product formation from the various 4-substituted phenylalanines.