The effect of phosphorylation on the shape of tyrosine hydroxylase (TH) was studied directly using gel filtration and indirectly using electrospray ionization mass spectrometry. Phosphorylation of Ser19 and Ser40 produced a TH molecule with a more open conformation than the non-phosphorylated form. The conformational effect of Ser19 phosphorylation is less pronounced than that of the Ser40 phosphorylation. The effect of Ser19 and Ser40 phosphorylation appears to be additive. Binding of dopamine produced a more compact form when compared with the non-dopamine-bound TH. The interdependence of Ser19 and Ser40 phosphorylation was probed using electrospray ionization mass spectrometry. The rate constants for the phosphorylation of Ser19 and Ser40 were determined by electrospray ionization mass spectrometry using a consecutive reaction model. The rate constant for the phosphorylation of Ser40 is ~2- to 3-fold higher if Ser19 is already phosphorylated. These results suggest that phosphorylation of Ser19 alters the conformation of tyrosine hydroxylase to allow increased accessibility of Ser40 to kinases.
Journal of Biological Chemistry Vol. 276, Issue 44, p. 40411-40416