Kinetic and mechanistic studies of a cell cycle protein phosphatase Cdc14

Wei Qing Wang, Joshua Bembenek, Kyle R. Gee, Hongtao Yu, Harry Charbonneau, Zhong Yin Zhang

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)30459-30468
Number of pages10
JournalJournal of Biological Chemistry
Volume279
Issue number29
DOIs
StatePublished - Jul 16 2004

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Cell Cycle Proteins
Phosphoprotein Phosphatases
Hydrolysis
Catalysis
Kinetics
Acids
Substrates
Phosphates
Cytokinesis
Cyclin-Dependent Kinases
Eukaryota
Phosphoric Monoester Hydrolases
Catalytic Domain
Proteins
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Wang, W. Q., Bembenek, J., Gee, K. R., Yu, H., Charbonneau, H., & Zhang, Z. Y. (2004). Kinetic and mechanistic studies of a cell cycle protein phosphatase Cdc14. Journal of Biological Chemistry, 279(29), 30459-30468. https://doi.org/10.1074/jbc.M402217200

Kinetic and mechanistic studies of a cell cycle protein phosphatase Cdc14. / Wang, Wei Qing; Bembenek, Joshua; Gee, Kyle R.; Yu, Hongtao; Charbonneau, Harry; Zhang, Zhong Yin.

In: Journal of Biological Chemistry, Vol. 279, No. 29, 16.07.2004, p. 30459-30468.

Research output: Contribution to journalArticle

Wang, WQ, Bembenek, J, Gee, KR, Yu, H, Charbonneau, H & Zhang, ZY 2004, 'Kinetic and mechanistic studies of a cell cycle protein phosphatase Cdc14', Journal of Biological Chemistry, vol. 279, no. 29, pp. 30459-30468. https://doi.org/10.1074/jbc.M402217200
Wang, Wei Qing ; Bembenek, Joshua ; Gee, Kyle R. ; Yu, Hongtao ; Charbonneau, Harry ; Zhang, Zhong Yin. / Kinetic and mechanistic studies of a cell cycle protein phosphatase Cdc14. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 29. pp. 30459-30468.
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