Identification by hydrogen/deuterium exchange of structural changes in tyrosine hydroxylase associated with regulation.
The activity of tyrosine hydroxylase is regulated by reversible phosphorylation of serine residues in an N-terminal regulatory domain and catecholamine inhibition at the active site. Catecholamines such as dopamine bind very tightly to the resting enzyme; phosphorylation of Ser40 decreases the affinity for catecholamines by 3 orders of magnitude. The effects of dopamine binding and phosphorylation of Ser40 on the kinetics of deuterium incorporation into peptide bonds were examined by mass spectrometry. When dopamine is bound, three peptic peptides show significantly slower deuterium incorporation, 35-41 and 42-71 in the regulatory domain and 295-299 in the catalytic domain. In the phosphorylated enzyme, peptide 295-299 shows more rapid incorporation of deuterium, while 35-41 and 42-71 can not be detected. These results are consistent with tyrosine hydroxylase existing in two different conformations. In the closed conformation, the regulatory domain lies across the active site loop containing residues 295-298; this is stabilized when dopamine is bound in the active site. In the open conformation, the regulatory domain has moved out of the active site, allowing substrate access; this conformation is favored by phosphorylation of Ser40.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Shanzhi
S
Wang
Giri R
GR
Sura
Lawrence J
LJ
Dangott
Paul F
PF
Fitzpatrick
D000818:Animals; D020134:Catalytic Domain; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D003903:Deuterium / Q000737:chemistry*; D041961:Deuterium Exchange Measurement / Q000379:methods; D004298:Dopamine / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000378:metabolism*
D002395:Catecholamines; D010446:Peptide Fragments; D012694:Serine; D006859:Hydrogen; D003903:Deuterium; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine