title: Cooperativity and evolution of Tetrahymena two-domain arginine kinase.
abstract: Tetrahymena pyriformis contains two arginine kinases, a 40-kDa enzyme (AK1) with a myristoylation signal sequence at the N-terminus and a two-domain 80-kDa enzyme (AK2). The former is localized mainly in cilia and the latter is in the cytoplasm. AK1 was successfully synthesized using an insect cell-free protein synthesis system and subjected to peptide mass fingerprinting (PMF) analysis. The masses corresponding to unmodified N-terminal tryptic peptide or N-terminal myristoylated peptide were not observed, suggesting that N-terminal peptides were not ionized in this analysis. We performed PMF analyses for two other phosphagen kinases (PKs) with myristoylation signals, an AK from Nematostella vectensis and a PK from Ectocarpus siliculosus. In both cases, the myristoylated, N-terminal peptides were clearly identified. The differences between the experimental and theoretical masses were within 0.0165-0.0583 Da, supporting the accuracy of the identification. Domains 1 and 2 of Tetrahymena two-domain AK2 were expressed separately in Escherichia coli and the extent of cooperativity was estimated on the basis of their kinetic constants. The results suggested that each of the domains functions independently, namely no cooperativity is displayed between the two domains. This is in sharp contrast to the two-domain AK from Anthopleura.
authors.affiliation: Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520 Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Okazaki
authors.affiliation: Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520 Japan.
authors.forename: Shou
authors.initials: S
authors.lastname: Motomura
authors.affiliation: Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520 Japan.
authors.forename: Nanaka
authors.initials: N
authors.lastname: Okazoe
authors.affiliation: Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520 Japan.
authors.forename: Daichi
authors.initials: D
authors.lastname: Yano
authors.affiliation: Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520 Japan. Electronic address: suzuki@kochi-u.ac.jp.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001122:Arginine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D015870:Gene Expression; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D000459:Phaeophyceae / Q000737:chemistry / Q000145:classification / Q000201:enzymology / Q000235:genetics; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012615:Sea Anemones / Q000737:chemistry / Q000145:classification / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013769:Tetrahymena pyriformis / Q000737:chemistry* / Q000145:classification / Q000201:enzymology / Q000235:genetics
chemical_list: D007527:Isoenzymes; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D001122:Arginine Kinase
keywords: Myristoylation; Phosphagen kinase; Two-domain enzyme