MUTATIONAL ANALYSES OF THE METAL-ION AND SUBSTRATE-BINDING SITES OF PHOSPHORYLASE-KINASE GAMMA-SUBUNIT

Phosphorylase kinase (PhK) and truncated gamma subunit, denoted gamma( 1-300), can phosphorylate seryl and tyrosyl residues dependent on the metal ion [Yuan, C.-J., Huang, C. F., & Graves, D. J. (1993) J. Biol. Chem. 268, 17683-17686]. Recombinant gamma(1-300) was used to explore its dual specificity and the location of the metal ion binding sites b y using site-directed mutagenesis. Two approaches were taken to genera te 26 mutants. First, on the basis of the crystal structure of cAMP-de pendent protein kinase (cAPK), the invariant Asn(155) and highly conse rved Asp(168)-Phe(169)-Gly(170) residues were mutated, Changes include d production of N155H, D168E, D168N, F169R, G170V, G170I, G170L (less than 1% of enzymatic activities were found in these mutants), F169W, a nd G170A mutants. Second, charge to alanine and charge reversal scanni ng mutations were used to probe the metal ion binding sites. Two mutan ts, E111K and E154R, showed very different metal ion response compared to wild-type gamma and were further characterized. The mutants F169W, G170A, E111K, and E154R had 15%, 5%, 8%, and 25% specific activity re lative to wild-type gamma, respectively. The folding pattern of wild-t ype and mutated enzyme forms of gamma was determined by photoacoustic infrared spectroscopy. Conformational disruptions were found in G170V, G170I, and G170L mutants, but the conformation of the rest of the mut ants was similar to that of wild-type gamma, suggesting that the loss of enzymatic activities of these mutants was not because of incorrect refolding. Kinetic analyses of mutants indicate that Asn(155) and Asp( 168) residues influence maximal velocity and that Glu(111) Glu(154), P he(169), and Gly(170) residues influence binding of MgATP and phosphor ylase b. The interactions of divalent cations, Mg2+ and Mn2+, with E11 1K, E154R, N155H, F169W, and G170A mutants were different from those w ith the wild-type, suggesting that Glu(111), Glu(154), Asn(155), Phe(1 69), and Gly(170) contribute to the character of the metal ion binding sites. Our results suggest that the metal ion binding sites reside be tween the D(168)FG loop and the E(111)-KPE(154)N loop, similar to the metal ion binding sites in cAPK. The tyrosine kinase activity of gamma (1-300) in the presence of Mn2+ was increased in E154R (251%), unchang ed in E111K, F169W, and G170A, and reduced in N155H (5%). Typically, t he mutations had a more pronounced effect on serine kinase activity th an on tyrosine kinase activity. The activity ratio (tyrosine kinase ac tivity to serine kinase activity) increased in the mutants, suggesting that these five residues have different roles in the two activities. Our results support the view that different conformational states indu ced by metal ions are important for dual specificity.