The Effect of Phosphorylation at Ser-40 on the Structure and Thermal Stability of Tyrosine Hydroxylase

Abstract

Several of the common multiprotein kinases phosphorylate one or more of these sites, and phosphorylation of Ser 31 and 40 is accompanied by significant enzyme activation in vitro . It has been proposed that activation of tyrosine hydroxylase (TH) by phosphorylation of Ser-40 is because of a conformational change that triggers the release of inhibitory catecholamines bound to the active site iron. The purpose of this chapter is to further investigate the conformational and stability changes induced by phosphorylation. Bovine adrenal TH (bTH) has been made isolated. The spectroscopic properties of the enzymes were examined before and after phosphorylation of Ser-40 by the catalytic subunit of bovine heart cyclic AMP (cAMP)-dependent protein kinase (PKA). The temperature-induced changes observed in the amide I band of the samples are similar to those reported previously, that is, a loss of regular secondary substructures and the emergence of band components at 1618 and 1685 cm -1 , which have been assigned to hydrogen-bonded extended structures formed upon aggregation of thermally denatured proteins. Therefore, protein unfolding can be followed by the temperature dependence of the intensity ratio of the amide I band at 1618 cm -1 and 1650 cm -1 . Phosphorylation has an opposite effect on these proteins; it decreases T m , for bTH by 4°C, whereas it increases its value by 3°C for human TH (hTH)1.