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 by using site-directed mutagenesis. Two approaches were taken to generate 26 mutants. First, on the basis of the crystal structure of cAMP-dependent protein kinase (cAPK), the invariant Asn155 and highly conserved Asp168-Phe169-Gly170 residues were mutated. Changes included production of N155H, D168E, D168N, F169R, G170V, G170I, G170L (less than 1% of enzymatic activities were found in these mutants), F169W, and G170A mutants. Second, charge to alanine and charge reversal scanning mutations were used to probe the metal ion binding sites. Two mutants, 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 relative to wild-type gamma, respectively. The folding pattern of wild-type 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 mutants was similar to that of wild-type gamma, suggesting that the loss of enzymatic activities of these mutants was not because of incorrect refolding.(ABSTRACT TRUNCATED AT 250 WORDS)
Huang
C Y
CY
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Yuan
C J
CJ
Luo
S
S
Graves
D J
DJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010764:Phosphorylase Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D013379:Substrate Specificity
D008670:Metals; D010446:Peptide Fragments; D010764:Phosphorylase Kinase; D011505:Protein-Tyrosine Kinases