title: Identification of amino acid residues responsible for taurocyamine binding in mitochondrial taurocyamine kinase from Arenicola brasiliensis.
abstract: In order to investigate the residues associated with binding of the substrate taurocyamine in Arenicola mitochondrial taurocyamine kinase (TK), we performed Ala-scanning of the amino acid sequence HTKTV at positions 67-71 on the GS loop, and determined apparent K(m) and V(max) (appK(m) and appV(max), respectively) of the mutant forms for the substrates taurocyamine and glycocyamine. The appK(m) values for taurocyamine of the K69A, T70A and V71A mutants were significantly increased as compared with wild-type, suggesting that these residues are associated with taurocyamine binding. Of special interest is a property of V71A mutant: its catalytic efficiency for glycocyamine was twice that for taurocyamine, indicating that the V71A mutant acts like a glycocyamine kinase, rather than a TK. The role of the amino acid residue K95 of Arenicola MiTK was also examined. K95 was replaced with R, H, Y, I, A and E. K95R, K95H and K95I have a 3-fold higher affinity for taurocyamine, and activity was largely lost in K95E. On the other hand, the K95Y mutant showed a rather unique feature; namely, an increase in substrate concentration caused a decrease in initial velocity of the reaction (substrate inhibition). This is the first report on the key amino acid residues responsible for taurocyamine binding in mitochondrial TK.
authors.affiliation: Kochi University, Kochi, Japan.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Tanaka
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Matsumoto
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D000595:Amino Acid Sequence / Q000502:physiology*; D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011077:Polychaeta / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013654:Taurine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D024101:Mitochondrial Proteins; D013654:Taurine; C009558:taurocyamine; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022716:taurocyamine kinase