Transition state analysis and requirement of Asp-262 general acid/base catalyst for full activation of dual-specificity phosphatase MKP3 by extracellular regulated kinase

Dual-specificity phosphatase MKP3 down-regulates mitogenic signaling throug h dephosphorylation of extracellular regulated kinase (ERK). Unlike a simpl e substrate-enzyme interaction, the noncatalytic, amino-terminal domain of MKP3 can bind efficiently to ERK, leading to activation of the phosphatase catalytic domain by as much as 100-fold toward exogenous substrates. It has been suggested that ERK activates MKP3 through the stabilization of the ac tive phosphatase conformation, enabling general acid catalysis. Here, we in vestigated whether Asp-262 of MKP3 is the bona fide general acid and evalua ted its contribution to the catalytic steps activated by ERK. Using site-di rected mutagenesis, pH rate and Bronsted analyses, kinetic isotope effects, and steady-state and rapid reaction kinetics, Asp-262 was identified as th e authentic general acid catalyst, donating a proton to the leaving group o xygen during P-O bond cleavage. Kinetic isotope effects [(18)(V/K)bridge, ( 18)(V/K)(nonbridge), and (15)(V/K)] were evaluated for the effect of ERK an d of the D262N mutation on the transition state of the phosphoryl transfer reaction. The patterns of the three isotope effects for the reaction with n ative MKP3 in the presence of ERK are indicative of a reaction where the le aving group is protonated in the transition state, whereas in the D262N mut ant, the leaving group departs as the anion. Even without general acid cata lysis, the D262N mutant reaction is activated by ERK through increased phos phate affinity (similar to8-fold) and the partial stabilization of the tran sition state for phospho-enzyme intermediate formation (similar to4-fold). Based on these analyses, we estimate that dephosphorylation of phosphorylat ed ERK by the D262N mutant is > 1000-fold lower than by native, activated M KP3. Also, the kinetic results suggest that Asp-262 functions as a general base during thiol-phosphate intermediate hydrolysis.