Phenylalanine residues in the active site of tyrosine hydroxylase: Mutagenesis of Phe300 and Phe309 to alanine and metal ion-catalyzed hydroxylation of Phe300

Residues Phe300 and Phe309 of tyrosine hydroxylase are located in the activ e site in the recently described three-dimensional structure of the enzyme, where they have been proposed to play roles in substrate binding. Also bas ed on the structure, Phe300 has been reported to be hydroxylated due to a n aturally occurring posttranslational modification [Goodwill, K. E., Sabatie r, 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 acti vity than wild-type tyrosine hydroxylase in the production of DOPA, but ful l activity in the production of dihydropterin. The F300A protein shows a 2. 5-fold decrease in activity in the production of both DOPA and dihydropteri n. The K-6-(MPH4) value for F300A tyrosine hydroxylase is twice the wild-ty pe value. These results are consistent with Phe309 having a role in maintai ning 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 o nly occurs in the isolated catalytic domain after incubation with a large e xcess 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 t he presence of excess iron. These results establish that hydroxylation of P he300 is an artifact of the crystallography conditions and is not relevant to catalysis.