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A more recent version of this article appeared on December 1, 2000
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Papers In Press, published online ahead of print September 26, 2000
J. Biol. Chem, 10.1074/jbc.C000606200
Submitted on September 1, 2000
Revised on September 21, 2000
Accepted on September 26, 2000

Chemical Rescue of a Mutant Protein Tyrosine Kinase

Daniel M. Williams, Dongxia Wang, and Philip A. Cole

Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Corresponding Author: pcole{at}jhmi.edu

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 kcat for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is ~3000-fold smaller compared to that of wild type Csk, whereas the Km values for ATP and poly(Glu,Tyr) are only mildly affected. The kcat for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R, R318A is 100-fold greater than the kcat 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.


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