Effects of substitution at serine 40 of tyrosine hydroxylase on catecholamine binding.
Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate of dissociation of inhibitory catecholamines [Ramsey, A. J., and Fitzpatrick, P. F. (1998) Biochemistry 37, 8980-8986]. To probe the structural basis for this effect and to ascertain the ability of other amino acids to functionally replace serine and serine phosphate, the effects of replacement of Ser40 with other amino acids were determined. Only minor changes in the Vmax value and the Km values for tyrosine and tetrahydropterin were seen upon replacement of Ser40 with alanine, valine, threonine, aspartate, or glutamate, in line with the minor effects of phosphorylation on steady-state kinetic parameters. More significant effects were seen on the binding of dopamine and dihydroxyphenylalanine. The affinity of the S40T enzyme for either catecholamine was very similar to that of the wild-type enzyme, while the S40E enzyme was similar to the phosphorylated enzyme. The S40D enzyme had an affinity for DOPA comparable to the phosphorylated enzyme but a higher affinity for dopamine than the latter. With both catecholamines, the S40V and S40A enzymes showed intermediate levels of activation. The results suggest that the serine hydroxyl contributes to the stabilization of the catecholamine-inhibited enzyme. In addition, the S40E enzyme will be useful in further studies of the effects of multiple phosphorylation on tyrosine hydroxylase, while the alanine enzyme does not provide an accurate mimic of the unphosphorylated enzyme.
McCulloch
R I
RI
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.
Daubner
S C
SC
Fitzpatrick
P F
PF
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002395:Catecholamines / Q000378:metabolism*; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*
D002395:Catecholamines; D011994:Recombinant Proteins; D012694:Serine; D004295:Dihydroxyphenylalanine; D014446:Tyrosine 3-Monooxygenase