Phenylalanine Residues in the Active Site of Tyrosine Hydroxylase: Mutagenesis of Phe300 and Phe309 to Alanine and Metal Ion-Catalyzed Hydroxylation of Phe300

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Abstract
Residues Phe300 and Phe309 of tyrosine hydroxylase are located in the active site in the recently described three-dimensional structure of the enzyme, where they have been proposed to play roles in substrate binding. Also based on the structure, Phe300 has been reported to be hydroxylated due to a naturally occurring posttranslational modification [Goodwill, K. E., Sabatier, C., and Stevens, R. C. (1998) Biochemistry 37, 13437−13445]. Mutants of tyrosine hydroxylase with alanine substituted for Phe300 or Phe309 have now been purified and characterized. The F309A protein possesses 40% less activity than wild-type tyrosine hydroxylase in the production of DOPA, but full activity in the production of dihydropterin. The F300A protein shows a 2.5-fold decrease in activity in the production of both DOPA and dihydropterin. The K6-MPH4 value for F300A tyrosine hydroxylase is twice the wild-type value. These results are consistent with Phe309 having a role in maintaining the integrity of the active site, while Phe300 contributes less than 1 kcal/mol to binding tetrahydropterin. Characterization of Phe300 by MALDI-TOF mass spectrometry and amino acid sequencing showed that hydroxylation only occurs in the isolated catalytic domain after incubation with a large excess of 7,8-dihydropterin, DTT, and Fe2+. The modification is not observed in the untreated catalytic domain or in the full-length protein, even in the presence of excess iron. These results establish that hydroxylation of Phe300 is an artifact of the crystallography conditions and is not relevant to catalysis.