title: Structural and mutational analysis of amino acid residues involved in ATP specificity of Escherichia coli acetate kinase.
abstract: Acetate kinase (AK) generally utilizes ATP as a phosphoryl donor, but AK from Entamoeba histolytica (PPi-ehiAK) uses pyrophosphate (PPi), not ATP, and is PPi-specific. The determinants of the phosphoryl donor specificity are unknown. Here, we inferred 5 candidate amino acid residues associated with this specificity, based on structural information. Each candidate residue in Escherichia coli ATP-specific AK (ATP-ecoAK), which is unable to use PPi, was substituted with the respective PPi-ehiAK amino acid residue. Each variant ATP-ecoAK had an increased Km for ATP, indicating that the 5 residues are the determinants for the specificity to ATP in ATP-ecoAK. Moreover, Asn-337 of ATP-ecoAK was shown to be particularly significant for the specificity to ATP. The 5 residues are highly conserved in 2625 PPi-ehiAK homologs, implying that almost all organisms have ATP-dependent, rather than PPi-dependent, AK.
authors.affiliation: Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Yoshioka
authors.affiliation: Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Kousaku
authors.initials: K
authors.lastname: Murata
authors.affiliation: Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan. Electronic address: kawais@kais.kyoto-u.ac.jp.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Kawai
mesh_terms: D000084:Acetate Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D011756:Diphosphates / Q000378:metabolism; D004748:Entamoeba histolytica / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013379:Substrate Specificity
chemical_list: D011756:Diphosphates; C107241:diphosphoric acid; D000255:Adenosine Triphosphate; D000084:Acetate Kinase
keywords: ATP; Acetate kinase; Entamoeba histolytica; Methanosarcina thermophila; Pyrophosphate