Chemical approaches to the study of protein tyrosine kinases and their implications for mechanism and inhibitor design. Review uri icon

Overview

MeSH

  • Phosphorylation
  • Protein Kinase Inhibitors
  • src-Family Kinases

MeSH Major

  • Chemistry, Pharmaceutical
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
  • Protein Kinases
  • Proteins

abstract

  • 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 Brønsted 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 Brønsted 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.

publication date

  • May 1999
  • June 1999

has subject area

  • Chemistry, Pharmaceutical
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
  • Phosphorylation
  • Protein Kinase Inhibitors
  • Protein Kinases
  • Proteins
  • src-Family Kinases

Research

keywords

  • Journal Article
  • Review

Identity

Language

  • eng

PubMed ID

  • 10454199

Additional Document Info

start page

  • 219

end page

  • 229

volume

  • 82

number

  • 2-3