A flexible loop in tyrosine hydroxylase controls coupling of amino acid hydroxylation to tetrahydropterin oxidation.
The role of a polypeptide loop in tyrosine hydroxylase (TyrH) whose homolog in phenylalanine hydroxylase (PheH) takes on a different conformation when substrates are bound has been studied using site-directed mutagenesis. The loop spans positions 177 to 191; alanine was introduced into those positions, introducing one alanine substitution per TyrH variant. Mutagenesis of residues in the center of the loop resulted in alterations in the KM values for substrates, the Vmax value for dihydroxyphenylalanine (DOPA) synthesis, and the coupling of tetrahydropterin oxidation to tyrosine hydroxylation. The variant with the most altered KM value for 6-methyltetrahydropterin was TyrH F184A. The variants with the most affected K(tyr) values were those with substitutions in the center of the loop, TyrH K183A, F184A, D185A, P186A and D187A. These five variants also had the most reduced Vmax values for DOPA synthesis. Alanine substitution in positions 182-186 resulted in lowered ratios of tyrosine hydroxylation to tetrahydropterin oxidation. TyrH F184Y and PheH Y138F, variants with the residue at the center of the loop substituted with the residue present at the homologous position in the other hydroxylase, were also studied. The V/K(tyr) to V/K(phe) ratios for these variants were altered significantly, but the results did not suggest that F184 of TyrH or Y138 of PheH plays a dominant role in determining amino acid substrate specificity.
Daubner
S Colette
SC
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.
McGinnis
James Thomas
JT
Gardner
Meredith
M
Kroboth
Stacie L
SL
Morris
Adam R
AR
Fitzpatrick
Paul F
PF
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011622:Pterins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D011622:Pterins; C011552:tetrahydropterin; D014443:Tyrosine; D010651:Phenylalanine Hydroxylase; D014446:Tyrosine 3-Monooxygenase