Abstract
PP2C family phosphatases (the type 2C family of protein phosphatases; or metal-dependent phosphatase, PPM) constitute an important class of signaling enzymes that regulate many fundamental life activities. All PP2C family members have a conserved binuclear metal ion active center that is essential for their catalysis. However, the catalytic role of each metal ion during catalysis remains elusive. In this study, we discovered that mutations in the structurally buried D38 residue of PP2Cα (PPM1A) redefined the water-mediated hydrogen network in the active site and selectively disrupted M2 metal ion binding. Using the D38A and D38K mutations of PP2Cα as specific tools in combination with enzymology analysis, our results demonstrated that the M2 metal ion determines the rate-limiting step of substrate hydrolysis, participates in dianion substrate binding and stabilizes the leaving group after P-O bond cleavage. The newly characterized catalytic role of the M2 metal ion in this family not only provides insight into how the binuclear metal centers of the PP2C phosphatases are organized for efficient catalysis but also helps increase our understanding of the function and substrate specificity of PP2C family members.
Highlights
PP2C family phosphatases constitute an important class of signaling enzymes that regulate many fundamental life activities
Using the D38A and D38K mutations of PP2Ca as specific tools in combination with enzymology analysis, our results demonstrated that the M2 metal ion determines the rate-limiting step of substrate hydrolysis, participates in dianion substrate binding and stabilizes the leaving group after P-O bond cleavage
The data shows a representative result from at least three experiments. (d) Statistical graph of Fig.2c. *: P, 0.05, cells transfected with PP2Ca-WT compared with control cells. #: P, 0.05, cells transfected with D38A or D38K compared with cells transfected with PP2Ca-WT
Summary
PP2C family phosphatases (the type 2C family of protein phosphatases; or metal-dependent phosphatase, PPM) constitute an important class of signaling enzymes that regulate many fundamental life activities. PKB (Akt) is a kind of serine/threonine-specific protein kinases and plays a key role in multiple cellular processes such as cell proliferation, apoptosis, cell migration and transcription Despite their distinct functions in cells, all PP2C family members have conserved active sites and share similar catalytic mechanisms. The essential roles of the binuclear center in PP2C family member activity have been confirmed by several biochemical and crystallographic analysis, two central, mutually linked questions regarding the catalysis of PP2C family members remain unanswered. Why do these phosphatases need two metal ions? Our study provides the exact roles of the M2 metal ion during catalysis, which will help in understanding the catalytic properties of PP2C family members and extend our knowledge about metal-mediated metalloenzyme catalysis
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