TY - JOUR
T1 - Chemical approaches to the study of protein tyrosine kinases and their implications for mechanism and inhibitor design
AU - Cole, Philip A.
AU - Sondhi, Dolan
AU - Kim, Kyonghee
N1 - Funding Information:
We thank M. Eck, W. Xu, Z. Songyang, and T. Muir for their key contributions to the studies described in this review. P. A. C. thanks the NIH, the Damon Runyon Scholar Award Program, the Irving Hansen Foundation, the Irma Hirschl Monique Weill-Caulier Trust, and the Burroughs Wellcome Toxicology New Investigator Award Program for generous support. K. K. is grateful for support from the Winston Foundation.
PY - 1999/5
Y1 - 1999/5
N2 - Protein tyrosine kinases are critical enzymes for signal transduction. Using C-terminal Src kinase (Csk) as a model system, we discuss progress in three main areas. First, we describe our efforts to measure the transition state of the reaction using peptide substrates containing fluorotyrosine analogs. It is shown that the Bronsted nucleophile coefficient for the reaction is near zero (similar to the nonenzymatic reaction) and the required nucleophile is the neutral phenol (rather than the more chemically reactive phenoxide anion). By studying the kinase reaction in the reverse direction, a Bronsted leaving group coefficient of -0.3 was measured, indicative of protonation of the departing phenol in the transition state. Taken together, these results strongly support a dissociative transition state mechanism for the kinase. These findings set constraints on the design of transition state analog inhibitors. Second, we describe efforts toward defining the specificity of Csk for peptide and protein substrates. The main findings are that local amino acids surrounding a phosphorylated tyrosine can influence recognition, but that long-range interactions probably are more important in a physiologic protein substrate. These findings underscore the complexities in how protein kinases select protein substrates. Third, we describe a new method in protein engineering that has been applied to the study of protein kinases. The method, expressed protein ligation, allows a general approach for ligating synthetic peptides to recombinant proteins. Using expressed protein ligation, obtaining site-specifically phosphorylated proteins and proteins with the incorporation of biophysical probes becomes relatively straightforward. We have used this method to generate a tail phosphorylated, conformationally altered Csk that showed an unexpected increase in kinase activity. Copyright (C) 1999 Elsevier Science Inc.
AB - Protein tyrosine kinases are critical enzymes for signal transduction. Using C-terminal Src kinase (Csk) as a model system, we discuss progress in three main areas. First, we describe our efforts to measure the transition state of the reaction using peptide substrates containing fluorotyrosine analogs. It is shown that the Bronsted nucleophile coefficient for the reaction is near zero (similar to the nonenzymatic reaction) and the required nucleophile is the neutral phenol (rather than the more chemically reactive phenoxide anion). By studying the kinase reaction in the reverse direction, a Bronsted leaving group coefficient of -0.3 was measured, indicative of protonation of the departing phenol in the transition state. Taken together, these results strongly support a dissociative transition state mechanism for the kinase. These findings set constraints on the design of transition state analog inhibitors. Second, we describe efforts toward defining the specificity of Csk for peptide and protein substrates. The main findings are that local amino acids surrounding a phosphorylated tyrosine can influence recognition, but that long-range interactions probably are more important in a physiologic protein substrate. These findings underscore the complexities in how protein kinases select protein substrates. Third, we describe a new method in protein engineering that has been applied to the study of protein kinases. The method, expressed protein ligation, allows a general approach for ligating synthetic peptides to recombinant proteins. Using expressed protein ligation, obtaining site-specifically phosphorylated proteins and proteins with the incorporation of biophysical probes becomes relatively straightforward. We have used this method to generate a tail phosphorylated, conformationally altered Csk that showed an unexpected increase in kinase activity. Copyright (C) 1999 Elsevier Science Inc.
KW - Expressed protein ligation
KW - Inhibition
KW - Mechanism
KW - Protein kinases
KW - Selectivity
KW - Transition state
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U2 - 10.1016/S0163-7258(98)00046-1
DO - 10.1016/S0163-7258(98)00046-1
M3 - Article
C2 - 10454199
AN - SCOPUS:0032987760
SN - 0163-7258
VL - 82
SP - 219
EP - 229
JO - Pharmacology and Therapeutics
JF - Pharmacology and Therapeutics
IS - 2-3
ER -