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

Holly R. Ellis, S. Colette Daubner, Ruth I. McCulloch, Paul F Fitzpatrick

Research output: Contribution to journalArticlepeer-review

Abstract

Residues PheSOO 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.

Original languageEnglish (US)
Pages (from-to)10913-10914
Number of pages2
JournalBiochemistry
Volume38
Issue number34
StatePublished - 1999
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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