Oxidation and site-directed mutagenesis of the sulfhydryl groups of a truncated γ catalytic subunit of phosphorylase kinase. Functional and structural effects

Chiun-Jye Yuan, C. Y.F. Huang, D. J. Graves*

*Corresponding author for this work

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

A truncated form of the γ subunit of phosphorylase kinase is inactivated by Cu2+ with the formation of two intra-molecular disulfide bonds. The formation of a disulfide bond between Cys-36 and Cys-172 (semioxidized form) results in approximately 50% loss of specific activity because the K(m) for MgATP is about 10-fold higher. The second disulfide bond is between Cys-184 and Cys-197 and causes further loss of activity. Eight Cys mutants, i.e. C36S, C36A, C42S, C138S, C172S, C184S, C184A, and C197S, were expressed and purified. Kinetic studies suggest that 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 kinase activity. The loss of tyrosine kinase activity is related to a lowered activity with Mn2+. With Mn2+, angiotensin II is a competitive inhibitor with respect to seryl kinase activity of C184S. With Mg2+, however, angiotensin II is a noncompetitive inhibitor. We suggest that metal ions influence the conformation of truncated γ and that the protein substrate binding region containing Cys-184 is important for the dual specificity of this kinase.

Original languageEnglish
Pages (from-to)24367-24373
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number39
StatePublished - 1 Jan 1994

Fingerprint Dive into the research topics of 'Oxidation and site-directed mutagenesis of the sulfhydryl groups of a truncated γ catalytic subunit of phosphorylase kinase. Functional and structural effects'. Together they form a unique fingerprint.

  • Cite this