PubMed:26260792 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PubMed/sourceid/26260792","sourcedb":"PubMed","sourceid":"26260792","source_url":"http://www.ncbi.nlm.nih.gov/pubmed/26260792","text":"Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A.\nThe 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 Mg(2+) 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.","tracks":[{"project":"sentences","denotations":[{"id":"T1","span":{"begin":0,"end":139},"obj":"Sentence"},{"id":"T2","span":{"begin":140,"end":299},"obj":"Sentence"},{"id":"T3","span":{"begin":300,"end":457},"obj":"Sentence"},{"id":"T4","span":{"begin":458,"end":619},"obj":"Sentence"},{"id":"T5","span":{"begin":620,"end":758},"obj":"Sentence"},{"id":"T6","span":{"begin":759,"end":963},"obj":"Sentence"},{"id":"T7","span":{"begin":964,"end":1161},"obj":"Sentence"},{"id":"T8","span":{"begin":1162,"end":1313},"obj":"Sentence"},{"id":"T9","span":{"begin":1314,"end":1562},"obj":"Sentence"},{"id":"T10","span":{"begin":1563,"end":1821},"obj":"Sentence"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/ontology/tao.owl#"}],"attributes":[{"subj":"T1","pred":"source","obj":"sentences"},{"subj":"T2","pred":"source","obj":"sentences"},{"subj":"T3","pred":"source","obj":"sentences"},{"subj":"T4","pred":"source","obj":"sentences"},{"subj":"T5","pred":"source","obj":"sentences"},{"subj":"T6","pred":"source","obj":"sentences"},{"subj":"T7","pred":"source","obj":"sentences"},{"subj":"T8","pred":"source","obj":"sentences"},{"subj":"T9","pred":"source","obj":"sentences"},{"subj":"T10","pred":"source","obj":"sentences"}]},{"project":"NCBITAXON","denotations":[{"id":"T1","span":{"begin":94,"end":107},"obj":"OrganismTaxon"},{"id":"T2","span":{"begin":397,"end":410},"obj":"OrganismTaxon"}],"attributes":[{"id":"A1","pred":"db_id","subj":"T1","obj":"5782"},{"id":"A2","pred":"db_id","subj":"T2","obj":"5782"},{"subj":"T1","pred":"source","obj":"NCBITAXON"},{"subj":"T2","pred":"source","obj":"NCBITAXON"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"sentences","color":"#caec93","default":true},{"id":"NCBITAXON","color":"#ec93e4"}]}]}}