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 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.
authors.affiliation: From the Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Yidai
authors.initials: Y
authors.lastname: Yang
authors.affiliation: From the Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Qilu
authors.initials: Q
authors.lastname: Ye
authors.affiliation: From the Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada jia@queensu.ca.
authors.forename: Zongchao
authors.initials: Z
authors.lastname: Jia
authors.affiliation: From the Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada coteg@queensu.ca.
authors.forename: Graham P
authors.initials: GP
authors.lastname: Côté
mesh_terms: D000227:Adenine Nucleotides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000227:Adenine Nucleotides; D015800:Protozoan Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase
keywords: adenosine; aspartyl phosphate; atypical protein kinase; catalysis; enzyme kinetics; myosin-II heavy chain kinase; protein kinase; x-ray crystallography; α-kinase