Identification of electrostatic interactions that determine the phosphorylation site specificity of the cAMP-dependent protein kinase.
"Charged-to alanine" scanning mutagenesis of the catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase (C1) identified three glutamate residues, E171, E214, and E274, that are involved in the recognition of a peptide substrate, kemptide (Leu1Arg2Arg3Ala4Ser5Leu6Gly7). These glutamate residues are conserved or conservatively substituted with asparate in the serine/threonine protein kinases that have a requirement for basic residues on the N-terminal side of their phosphorylation sites. Alanine replacement mutants in C1 were subjected to kinetic analysis using alanine-substituted peptides as substrates. The additivity or nonadditivity of the effects of the alanine substitutions on the catalytic efficiency (kcat/Km) was analyzed. This allowed the identification of electrostatic interactions between the three glutamate residues in the enzyme and the two arginine residues present in the peptide substrate. The data suggest that E171 interacts with Arg2 in the substrate and that E214 and E274 both interact with Arg3. This may be a general method for identifying simple intermolecular interactions involving proteins when there is no three-dimensional structure available of the complex of interacting species. The identification of these interactions provides the potential for rational protein engineering of enzymes with alternative specificities.
Gibbs
C S
CS
Department of Protein Engineering, Genentech Inc., South San Francisco, California 94080.
Zoller
M J
MJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D005971:Glutamates / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D005971:Glutamates; D009842:Oligopeptides; C037642:kemptide; D011494:Protein Kinases