The transfer of phosphoryl groups from ATP to serine and tyrosine residues in target proteins in the eucaryotic cell is catalyzed by a large class of enzymes known as protein kinases. The relevance of these enzymes in proper cell function is underscored by the observation that mutant forms of these catalysts are associated with uncontrolled cell growth and tumor formation. While a significant effort has recently been placed on the structural elucidation of several protein kinases, much less is known about the kinetics of phosphoryl group transfer and the participation of amino acid residues that facilitate this transfer. The goal of this proposal is to define the kinetic differences between serine and tyrosine phosphorylation catalyzed by serine and tyrosine protein kinases. To achieve this goal, we have selected two model systems- the kinase domain of the oncogenic nonreceptor tyrosine protein kinase, v-fps, and the catalytic subunit of the serine protein kinase, cAMP-dependent protein kinase. Both enzymes are well expressed in E. coli and are readily amenable to detailed kinetic analyses. The results of these studies will be used to support the hypothesis that serine and tyrosine phosphorylation occur by similar kinetic mechanisms that differ only by the rate of phosphoryl group transfer. We will demonstrate that the rates of substrate and product binding and any conformational changes associated with these processes are highly similar. We will also demonstrate that both enzymes employ general-base catalytic mechanisms to achieve rapid phosphoryl group transfer and that the faster rates of serine versus tyrosine phosphorylation are due to positioning of the g phosphoryl group of ATP, a phenomenon that is controlled indirectly through autophosphorylation. We will prove our hypothesis by achieving four specific objectives. First, we will determine complete kinetic mechanisms for the two kinase domains that include the rate constants for phosphoryl group transfer and substrate and product binding using rapid quench flow kinetic and solvent perturbation techniques. Second, we will determine whether these enzymes employ general-base catalysis for phosphoryl group transfer using pH-dependent steady-state and pre-steady-state kinetic and mutagenesis methods. Third, we will determine the substrate analogs. Fourth, we will determine the role of autophosphorylation site for substrate processing in v-fps and compare it to that for cAPK using mutagenesis and kinetic methods.
