OXIDATION AND SITE-DIRECTED MUTAGENESIS OF THE SULFHYDRYL-GROUPS OF ATRUNCATED GAMMA-CATALYTIC SUBUNIT OF PHOSPHORYLASE-KINASE - FUNCTIONAL AND STRUCTURAL EFFECTS

A truncated form of the gamma subunit of phosphorylase kinase is inact ivated by CU2+ with the formation of two intra-molecular disulfide bon ds. The formation of a disulfide bond between Cys-36 and Cys-172 (semi oxidized form) results in approximately 50% loss of specific activity because the K-m for MgATP is about 10-fold higher. The second disulfid e bond is between Cys-184 and Cys-197 and causes further loss of activ ity. Eight Cys mutants, i.e. C36S, C36A, C42S, C138S, C172S, C184S, C1 84A, and C197S, were expressed and purified. Kinetic studies suggest t hat Cys-36 is important for interaction at the nucleotide site because of its hydrophobicity. With Cys-184 mutants, C184S and C184A, tyrosyl phosphorylation of angiotensin II is affected much more than serine k inase activity. The loss of tyrosine kinase activity is related to a l owered activity with Mn2+. With Mn2+, angiotensin II is a competitive inhibitor with respect to seryl kinase activity of C184S. With Mg2+, h owever, angiotensin II is a noncompetitive inhibitor. We suggest that metal ions influence the conformation of truncated gamma and that the protein substrate binding region containing Cys-184 is important for t he dual specificity of this kinase.