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:

Yang, Yidai ^[1]; Ye, Qilu ^[1]; Jia, Zongchao ^[1]; Côté, Graham P. ^[1]

---

+ Show Author Affiliations

1. Queen's Univ., Kingston, ON (Canada)

Publication Date:

2015-08-10

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