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 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 K(6-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 Fe(2+). 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.
Ellis
H R
HR
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
Daubner
S C
SC
McCulloch
R I
RI
Fitzpatrick
P F
PF
D000409:Alanine / Q000235:genetics*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D006900:Hydroxylation; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D010649:Phenylalanine; D007501:Iron; D014446:Tyrosine 3-Monooxygenase; D000409:Alanine