Mutagenesis of a Specificity-Determining Residue in Tyrosine Hydroxylase Establishes That the Enzyme Is a Robust Phenylalanine Hydroxylase but a Fragile Tyrosine Hydroxylase

Abstract

The aromatic amino acid hydroxylases tyrosine hydroxylase (TyrH) and phenylalanine hydroxylase (PheH) have essentially identical active sites, but PheH is nearly incapable of hydroxylating tyrosine, while TyrH can readily hydroxylate both tyrosine and phenylalanine. Previous studies have indicated that Asp425 of TyrH is important in determining the substrate specificity of that enzyme (Daubner, S. C., Melendez, J., and Fitzpatrick, P. F. (2000) Biochemistry 39, 9652–9661). Alanine scanning mutagenesis of amino acids 423-427, a mobile loop containing Asp425, shows that only mutagenesis of Asp425 alters the activity of the enzyme significantly. Saturation mutagenesis of Asp425 results in large (up to 104) decreases in the Vmax and Vmax/Ktyr values for tyrosine hydroxylation, but only small decreases or even increases in the Vmax and Vmax/Kphe values for phenylalanine hydroxylation. The decrease in the tyrosine hydroxylation activity of the mutant proteins is due to an uncoupling of tetrahydropterin oxidation from amino acid hydroxylation with tyrosine as the amino acid substrate. In contrast, with the exception of the D425W mutant, the coupling of tetrahydropterin oxidation and amino acid hydroxylation is unaffected or increases with phenylalanine as the amino acid substrate. The decrease in the Vmax value with tyrosine as substrate shows a negative correlation with the hydrophobicity of the amino acid residue at position 425. The results are consistent with a critical role of Asp425 being to prevent a hydrophobic interaction that results in a restricted active site in which hydroxylation of tyrosine does not occur.

Functional Studies of Tyrosine Hydroxylase Missense Variants Reveal Distinct Patterns of Molecular Defects in Dopa ‐Responsive Dystonia

“…Asp‐425 in rat TH (corresponding to Asp‐455 in hTH4; Fig. ) has recently been shown to be a determinant for the substrate specificity in TH, by critically avoiding hydrophophic interactions that decrease the preference for l ‐Tyr without affecting the hydroxylation of l ‐Phe [Daubner et al., ]. Although the ability of TH to hydroxylate l ‐Phe in addition to l ‐Tyr could be considered an advantage in phenylketonuria (PKU; MIM #261600) where there is an excess of l ‐Phe and low availability of l ‐Tyr, the competitive nature of these substrates could also compromise tyrosine hydroxylase activity and catecholamine synthesis in this condition.…”

The Solution Structure of the Regulatory Domain of Tyrosine Hydroxylase

“…Mutagenesis of a single Asp residue in a flexible loop in TyrH can effectively abolish all activity as a TyrH while increasing its residual activity as a PheH. 58 …”

Neural versus alternative integrative systems: molecular insights into origins of neurotransmitters

“…Surprisingly, in TH, a single mutation of Asp425 (D425 V) nearly abolishes the enzymatic activity for the enzyme to produce L-DOPA [227]. And the relative specificity of TH for phenylalanine versus tyrosine, as measured by the (V/Kphe)/(V/ Ktyr) value, increased by 80 000-fold in the D425 V enzyme [228]. Only predicted TH enzymes contain this critical amino acid residue, and none of the non-bilaterian putative PAH contains this residue but contain nonpolar residues.…”