title: Chemical rescue of a mutant protein-tyrosine kinase.
abstract: Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase C-terminal Src kinase (Csk) was investigated. The observed k(cat) for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is approximately 3000-fold smaller compared with that of wild type Csk, whereas the K(m) values for ATP and poly(Glu,Tyr) are only mildly affected. The k(cat) value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the k(cat) value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Williams
authors.forename: D
authors.initials: D
authors.lastname: Wang
authors.forename: P A
authors.initials: PA
authors.lastname: Cole
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine; D001224:Aspartic Acid; D000081247:CSK Tyrosine-Protein Kinase; D020134:Catalytic Domain; D002645:Chickens; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D019061:src-Family Kinases
chemical_list: D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D001120:Arginine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases