Characterization of the Catalytic and Nucleotide Binding Properties of the Alpha-kinase domain of Dictyostelium Myosin-II Heavy Chain Kinase A. [Protein Structure and Folding]

August 10th, 2015 by Yang, Y., Ye, Q., Jia, Z., Cote, G. P.

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.
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