MUTATION OF POSITION-52 IN ERK2 CREATES A NONPRODUCTIVE BINDING MODE FOR ADENOSINE 5'-TRIPHOSPHATE

Among the protein kinases, an absolutely conserved lysine in subdomain II is required for high catalytic activity. This lysine is known to i nteract with the substrate ATP, but otherwise its role is not well und erstood. We have used biochemical and structural methods to investigat e the function of this lysine (K52) in phosphoryl transfer reactions c atalyzed by the MAP kinase ERK2. The kinetic properties of activated w ild-type ERK2 and K52 mutants were examined using the oncoprotein TAL2 , myelin basic protein, and a designed synthetic peptide as substrates . The catalytic activities of K52R and K52A ERK2 were lower than that of wild-type ERK2, primarily as a consequence of reductions in k(cat). Further, there was little difference in K-m for ATP, but the K-m,K-ap p for peptide substrate was higher for the K52 mutants. The three-dime nsional structure of unphosphorylated K52R ERK2 in the absence and pre sence of bound ATP was determined and compared with the structure of u nphosphorylated wild-type ERK2. ATP adopted a well-defined but distinc t binding mode in K52R ERK2 compared to the binding mode in the wild-t ype enzyme, The structural and kinetic data show that mutation of K52 created a nonproductive binding mode for ATP and suggest that K52 is e ssential for orienting ATP for catalysis.