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Publication Abstract from PubMed

The alpha-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 alpha-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 Asp766 residue. The results show that the beta- and alpha-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp766. 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-Methyl-anthraniloyl) analogs of ATP and ADP yielded Kd values for ATP, ADP, AMP and adenosine of 20, 60, 160 and 45 micromole, respectively. Site-directed mutagenesis showed that Glu713, Leu716 and Lys645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln758, which chelates a nucleotide-associated Mg2+ ion, eliminated catalytic activity, whereas loss of the highly conserved Lys722 and Arg592 decreased kcat for kinase and ATPase activities by 3-6-fold. Mutation of Asp663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln768 doubled ATPase activity, suggesting that it may act to exclude water from the active site.

Characterization of the Catalytic and Nucleotide Binding Properties of the Alpha-kinase domain of Dictyostelium Myosin-II Heavy Chain Kinase A.,Yang Y, Ye Q, Jia Z, Cote GP J Biol Chem. 2015 Aug 10. pii: jbc.M115.672410. PMID:26260792^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.