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Title: Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A

Abstract

The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with k cat values of 1.9, 0.6, and 0.32 min –1, respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2'/3'-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded K d values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μm, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg 2+ ion, eliminated catalytic activity, whereas loss of the highlymore » conserved Lys-722 and Arg-592 decreased k cat values for kinase and ATPase activities by 3–6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site.« less

Authors:
 [1];  [1];  [1];  [1]
  1. Queen's Univ., Kingston, ON (Canada)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
Natural Science and Engineering Research Council of Canada
OSTI Identifier:
1252766
Grant/Contract Number:  
RGPIN 391522; RGPIN 203705
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 290; Journal Issue: 39; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; adenosine; catalysis; enzyme kinetics; protein kinase; x-ray crystallography; α-kinase; aspartyl phosphate; atypical protein kinase; myosin-II heavy chain kinase

Citation Formats

Yang, Yidai, Ye, Qilu, Jia, Zongchao, and Côté, Graham P. Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A. United States: N. p., 2015. Web. doi:10.1074/jbc.M115.672410.
Yang, Yidai, Ye, Qilu, Jia, Zongchao, & Côté, Graham P. Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A. United States. https://doi.org/10.1074/jbc.M115.672410
Yang, Yidai, Ye, Qilu, Jia, Zongchao, and Côté, Graham P. Mon . "Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A". United States. https://doi.org/10.1074/jbc.M115.672410. https://www.osti.gov/servlets/purl/1252766.
@article{osti_1252766,
title = {Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A},
author = {Yang, Yidai and Ye, Qilu and Jia, Zongchao and Côté, Graham P.},
abstractNote = {The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with kcat values of 1.9, 0.6, and 0.32 min–1, respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2'/3'-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded Kd values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μm, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg2+ ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased kcat values for kinase and ATPase activities by 3–6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site.},
doi = {10.1074/jbc.M115.672410},
url = {https://www.osti.gov/biblio/1252766}, journal = {Journal of Biological Chemistry},
issn = {0021-9258},
number = 39,
volume = 290,
place = {United States},
year = {2015},
month = {8}
}

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