Abstract
The Cdc14 family of protein phosphatases is conserved within eukaryotes and antagonizes the action of cyclin-dependent kinases, thereby promoting mitotic exit and cytokinesis. We performed a detailed kinetic and mechanistic study of the Cdc14 phosphatases with both small molecule aryl phosphates and a physiological protein substrate hCdh1. We found that Cdc14 displays a strong preference for two-ringed aryl phosphates over smaller one-ringed or larger, multi-ringed substrates, a finding that may have important implications for inhibitor design. Results from both leaving group and pH dependence of the Cdc14-catalyzed reaction are consistent with a general acid-independent mechanism for substrates with leaving group pKa < 7 and a general acid-dependent mechanism for substrates with leaving group pKa > 7. The use of both low and high leaving group pKa substrates, in combination with steady-state and pre-steady-state kinetic techniques enabled the isolation and analysis of both the phosphoenzyme (E-P) formation and hydrolysis step. We established the requirement of general acid catalysis for E-P formation in reactions with high leaving group pKa substrates, and the presence of general base catalysis in E-P hydrolysis. Mutational study of invariant acidic residues in Cdc14 identified Asp253 as the general acid during E-P formation and the general base in E-P hydrolysis. We also identified several residues including Asp50, Asp129, Glu168, Glu171, and Asp177 in the Cdc14 active site cleft that are required for efficient dephosphorylation of hCdh1.
Highlights
Cdc14 is a protein phosphatase conserved from yeast to man [1, 2]
We identified several residues including Asp50, Asp129, Glu168, Glu171, and Asp177 in the Cdc14 active site cleft that are required for efficient dephosphorylation of human Cdh1 (hCdh1)
It is possible that Cdc14 phosphatases may antagonize cyclin-dependent kinase (CDK) activity by dephosphorylation of different substrates and regulate distinct cellcycle transitions in different species, as CDK activity is important for many different cell cycle processes [14]
Summary
Cdc14 is a protein phosphatase conserved from yeast to man [1, 2]. In the budding yeast, Saccharomyces cerevisiae, Cdc14 is essential for cell cycle progression from late anaphase into G1 of the subsequent cell cycle, a process referred to as exit from mitosis [3,4,5]. The fact that the Cdc14-catalyzed hydrolysis of pNPP and DiFMUP, two substrates that differ markedly in their leaving group pKa (7.14 for p-nitrophenol and 4.7 for DiFMU), display similar kcat values suggests that the rate-determining step is mostly E-P hydrolysis ( see below).
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