| The insulin receptor and many other protein kinases are activated by relief of intrasteric inhibition which is regulated by reversible autophosphorylation of tyrosines in the activation loop. Three aspects of kinase regulation through this motif have been addressed in this work. First, the conformational and catalytic-cycle changes accompanying activation of the insulin receptor's kinase domain are analyzed through steady-state kinetics, viscometric analysis, and fluorescence. The key changes involve the switch from a chemical step-limited reaction to a diffusive step-limited reaction. Thermodynamically, the increased catalytic efficiency originates from a nearly equal redistribution of conformational free energy released upon autophosphorylation of the kinase to improved substrate binding and to lowering the kinetic barrier between ternary complexes and the transition states.; Second, collaborative studies on the chemical mechanism of the enzyme-catalyzed phosphoryl transfer reaction using a tetrafluorotyrosyl-substrate and a bisubstrate analog support a dissociative reaction pathway for the transition state. Finally, using rationally designed single amino acid replacement mutagenesis, selected residues in the activation loop and conserved catalytic core were probed for their contributions to stabilizing the intrasterically inhibited basal state. This work reveals a range of activation loop conformations with correspondingly graded inhibitory properties that may uncover fundamental regulatory features shared among receptor tyrosine kinases. |
