Kinetic and mechanistic studies of a cell cycle protein phosphatase Cdc14.
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.
Wang
Wei-Qing
WQ
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Bembenek
Joshua
J
Gee
Kyle R
KR
Yu
Hongtao
H
Charbonneau
Harry
H
Zhang
Zhong-Yin
ZY
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000737:chemistry; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D017173:Caenorhabditis elegans; D002384:Catalysis; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009943:Organophosphorus Compounds / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D000814:Aniline Compounds; C074860:CDC14 protein, S cerevisiae; D018797:Cell Cycle Proteins; D009943:Organophosphorus Compounds; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; C068798:4-aminophenylphosphate; D017027:Protein Tyrosine Phosphatases