Oxidation and site-directed mutagenesis of the sulfhydryl groups of a truncated gamma catalytic subunit of phosphorylase kinase. Functional and structural effects.
A truncated form of the gamma 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 Km 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 gamma and that the protein substrate binding region containing Cys-184 is important for the dual specificity of this kinase.
Yuan
C J
CJ
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Huang
C Y
CY
Graves
D J
DJ
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000378:metabolism; D002384:Catalysis; D003300:Copper / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D000804:Angiotensin II; D003300:Copper; D010764:Phosphorylase Kinase; D003545:Cysteine