MUTATIONAL ANALYSIS OF THE NUCLEOTIDE-BINDING SITE OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR AND V-SRC PROTEIN-TYROSINE KINASES

Tyrosine kinases differ from serine/threonine kinases in sequences loc ated at the active site where ATP and substrate bind. In the structure of cyclic AMP-dependent protein kinase, the catalytic loop contains t he sequence Lys-Pro-Glu where the Lys residue contacts the gamma-phosp hate of ATP and the Glu residue contacts a basic residue located in th e peptide substrate. In tyrosine kinases, the analogous sequence is Al a-Ala-Arg in the receptor tyrosine kinase subfamily and Arg-Ala-Ala in the Src tyrosine kinase subfamily. To deduce the role of these residu es in tyrosine kinase function, site-directed mutations were prepared in the epidermal growth factor receptor (EGFR) and in v-Src and effect s on ATP binding and kinase activity were determined. Changing Arg to either Lys or Ala dramatically reduced activity of both tyrosine kinas es and this correlated with loss of ATP binding. Changing the orientat ion of this sequence impaired activity of EGFR to a greater extent tha n that of v-Src but did not change substrate specificity of the two en zymes. These results support the hypothesis that Arg functions to coor dinate the gamma-phosphate of ATP. Analysis of sequence inversions in the catalytic loop indicate that the active site of v-Src exhibits gre ater flexibility than that of EGFR.