TI - The mechanism of p21-activated kinase 2 autoactivation . AB - The p21-activated kinases ( PAKs ) play an important role in diverse cellular processes . PAK2 is activated by autophosphorylation upon binding of small G proteins such as Cdc42 and Rac in the GTP-bound state . However , the mechanism of PAK2 autophosphorylation in vitro is unclear . In the present study , the kinetic theory of the substrate reaction during modification of enzyme activity has been applied to a study of the autoactivation of PAK2 . On the basis of the kinetic equation of the substrate reaction during the autophosphorylation of PAK2 , the activation rate constants for the free enzyme and enzyme -substrate complex have been determined . The results indicate that 1 ) in the presence of Cdc42 , PAK2 autophosphorylation is a bipartite mechanism , with the regulatory domain autophosphorylated at multiple residues , whereas activation coincides with autophosphorylation of the catalytic domain at Thr-402 ; 2 ) the autophosphorylation reactions in regulatory domain are either a nonlimiting step or not required for activation of enzyme ; 3 ) the autophosphorylation at site Thr-402 on the catalytic domain occurs by an intermolecular mechanism and is required for phosphorylation of exogenous substrates examined ; 4 ) binding of the exogenous protein/peptide substrates at the active site of PAK2 has little or no effect on the autoactivation of PAK2 , suggesting that multiple regions of PAK2 are involved in the enzyme -substrate recognition . The present method also provides a novel approach for studying autophosphorylation reactions . Since the experimental conditions used resemble more closely the in vivo situation where the substrate is constantly being turned over while the enzyme is being modified , this new method would be particularly useful when the regulatory mechanisms of the reversible phosphorylation reaction toward certain enzymes are being assessed .