Effects of substitution at serine 40 of tyrosine hydroxylase on catecholamine binding

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase a ctivates the enzyme by increasing the rate of dissociation of inhibitory ca techolamines [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 V-max value and the K-m values for t yrosine and tetrahydropterin were seen upon replacement of Ser40 with alani ne, valine, threonine, aspartate, or glutamate, in line with the minor effe cts of phosphorylation on steady state kinetic parameters. More significant effects were seen on the binding of dopamine and dihydroxyphenylalanine. T he 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 pho sphorylated enzyme. The S40D enzyme had an affinity for DOPA comparable to the phosphorylated enzyme but a higher affinity for dopamine than the latte r. With both catecholamines, the S40V and S40A enzymes showed intermediate levels of activation. The results suggest that the serine hydroxyl contribu tes to the stabilization of the catecholamine-inhibited enzyme. In addition , the S40E enzyme will be useful in further studies of the effects of multi ple phosphorylation on tyrosine hydroxylase, while the alanine enzyme does not provide an accurate mimic of the unphosphorylated enzyme.