Kinetic analysis of human serine threonine protein phosphatase 2Cα

The PPM family of Ser/Thr protein phosphatases have recently been shown to down-regulate the stress response pathways in eukaryotes. Within the stress pathway, key signaling kinases, which are activated by protein phosphorylation, have been proposed as the in vivo substrates of PP2C, the prototypical member of the PPM family. Although it is known that these phosphatases require metal cations for activity, the molecular details of these important reactions have not been established. Therefore, here we report a detailed biochemical study to elucidate the kinetic and chemical mechanism of PP2C alpha. Steady-state kinetic and product inhibition studies revealed that PP2C alpha employs an ordered sequential mechanism, where the metal cations bind before phosphorylated substrate, and phosphate is the last product to be released. The metal-dependent activity of PP2C (as reflected in k(cat) and k(cat)/K-m), indicated that Fe2+ was 1000-fold better than Mg2+. The pH rate profiles revealed two ionizations critical for catalytic activity. An enzyme ionization with a pK(a) value of 7 must be unprotonated for catalysis, and an enzyme ionization with a pK(a) of 9 must be protonated for substrate binding. Bronsted analysis of substrate leaving group pK(a), indicated that phosphomonoester hydrolysis is rate-limiting at pH 7.0, but not at pH 8.5 where a common step independent of the nature of the substrate and alcohol product limits turnover (k(cat)). Rapid reaction kinetics between phosphomonoester and PP2C yielded exponential "bursts" of product formation, consistent with phosphate release being the slow catalytic step at pH 8.5. Dephosphorylation of synthetic phosphopeptides corresponding to several protein kinases revealed that PP2C displays a strong preference for diphosphorylated peptides in which the phosphorylated residues are in close proximity.