A basic residue, Lys 782, composes part of the ATP-binding site on the epidermal growth factor receptor tyrosine kinase

To identify amino acids specific for tyrosine kinase activity, the role of several conserved basic residues in kinase function was tested. Modeling of the epidermal growth factor receptor tyrosine kinase domain based on the c rystal structure of cyclic AMP-dependent protein kinase and insulin recepto r revealed several basic residues present on the surface of epidermal growt h factor receptor. Using the molecular modeling program, GRASP, the basic r esidues Arg 779, Lys 782, and Lys 855 were shown to provide an area of posi tive charge to the surface of the molecule, To deduce the role of these res idues in ATP and substrate binding, site-directed mutants were prepared and kinetic constants were measured. Mutation of Lys 855 to Ala destabilized t he enzyme and caused partial inactivation. Mutation of either Arg 779 or Ly s 782 had little effect on the K-m value for peptide substrate. However, al teration of Lys 782 increased the K-m value for ATP 28-fold, indicating a r ole for Lys 782 in binding ATP. Because residues similar to Lys 782 in the sequences of mitogen-activated protein kinase and insulin receptor make con tact with a ribose hydroxyl of ATP, it is proposed that Lys 782 may be one of the residues composing the ribose-binding site of epidermal growth facto r receptor. (C) 1999 Academic Press.