title: The role of Y84 on domain 1 and Y87 on domain 2 of Paragonimus westermani taurocyamine kinase: Insights on the substrate binding mechanism of a trematode phosphagen kinase.
abstract: The two-domain taurocyamine kinase (TK) from Paragonimus westermani was suggested to have a unique substrate binding mechanism. We performed site-directed mutagenesis on each domain of this TK and compared the kinetic parameters Km(Tc) and Vmax with that of the wild-type to determine putative amino acids involved in substrate recognition and binding. Replacement of Y84 on domain 1 and Y87 on domain 2 with R resulted in the loss of activity for the substrate taurocyamine. Y84E mutant has a dramatic decrease in affinity and activity for taurocyamine while Y87E has completely lost catalytic activity. Substituting H and I on the said positions also resulted in significant changes in activity. Mutation of the residues A59 on the GS region of domain 1 also caused significant decrease in affinity and activity while mutation on the equivalent position on domain 2 resulted in complete loss of activity.
authors.affiliation: Department of Environmental Health Sciences, Kochi University, Kochi 783-8505, Japan; Department of Immunology, Research Institute for Tropical Medicine, Muntinlupa 1781, Philippines.
authors.forename: Blanca R
authors.initials: BR
authors.lastname: Jarilla
authors.forename: Shinji
authors.initials: S
authors.lastname: Tokuhiro
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Nagataki
authors.forename: Kouji
authors.initials: K
authors.lastname: Uda
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Suzuki
authors.forename: Luz P
authors.initials: LP
authors.lastname: Acosta
authors.forename: Takeshi
authors.initials: T
authors.lastname: Agatsuma
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D048492:Paragonimus westermani / Q000201:enzymology*; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D013654:Taurine / Q000031:analogs & derivatives* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
chemical_list: D013654:Taurine; D014443:Tyrosine; C009558:taurocyamine; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022716:taurocyamine kinase
keywords: AK; CK; GK; LK; PK; Paragonimus westermani; Phosphagen kinase; Site-directed mutagenesis; TK; Taurocyamine kinase; Trematode; arginine kinase; creatine kinase; glycocyamine kinase; lombricine kinase; phosphagen kinase; taurocyamine kinase