Phosphagen kinase of the giant tubeworm Riftia pachyptila. Cloning and expression of cytoplasmic and mitochondrial isoforms of taurocyamine kinase.
The giant tubeworm Riftia pachyptila lives at deep-sea hydrothermal vents along the East Pacific Rise and the Galapagos Rift. The large size and high growth rate of R. pachyptila is supported by an endosymbiotic relationship with a chemosynthetic bacterium. Elucidation of the regulation of energy metabolism of the giant tubeworm remains an interesting problem. The purpose of this study is to determine the cDNA sequence of phosphagen kinase, one of the most important enzymes in energy metabolism, and to characterize its function. Two phosphagen kinase cDNA sequences amplified from the cDNA library of R. pachyptila showed high derived amino acid sequence identity (74%) with those of cytoplasmic taurocyamine kinase (TK) and mitochondrial TK from an annelid Arenicola brasiliensis. The cytoplasmic form of the Riftia recombinant enzyme showed stronger activity for the substrates taurocyamine and also considerable activity for lombricine (21% that of taurocyamine). The mitochondrial form, which was structurally similar to mitochondrial creatine kinase, showed stronger activity for taurocyamine, and a broader activity for various guanidine compounds: glycocyamine (35% that of taurocyamine), lombricine (31%) and arginine (3%). Both forms showed no activity for creatine. The difference in substrate specificities between the cytoplasmic and mitochondrial forms might be attributable to the large difference in the amino acid sequence of the GS region and/or several key amino acid residues for establishing guanidine substrate specificity. Based on these results, we conclude that Riftia contains at least two forms of TK as phosphagen kinase. We also report the kinetic parameters, Km and kcat, of Arenicola and Riftia TKs for the first time. The Km values for taurocyamine of Arenicola and Riftia TKs ranged from 0.9 to 4.0 mM and appear to be comparable to those of other annelid-specific enzymes, lombricine kinase and glycocyamine kinase, but are significantly lower than those of Neanthes cytoplasmic and mitochondrial creatine kinases. Comparison of kcat/Km value in various annelid phosphagen kinases indicates that Arenicola mitochondrial TK has the highest catalytic efficiency (16.2 s-1 mM-1). In Arenicola TKs, the mitochondrial form has seven-fold higher efficiency than the cytoplasmic form.
Uda
Kouji
K
Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520, Japan.
Tanaka
Kumiko
K
Bailly
Xavier
X
Zal
Franck
F
Suzuki
Tomohiko
T
D000595:Amino Acid Sequence; D000818:Animals; D000845:Annelida; D001120:Arginine / Q000737:chemistry; D002384:Catalysis; D003001:Cloning, Molecular; D003401:Creatine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D019143:Evolution, Molecular; D015723:Gene Library; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry; D006376:Helminths; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D010802:Phylogeny; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000031:analogs & derivatives / Q000737:chemistry; D013696:Temperature
D018076:DNA, Complementary; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012694:Serine; C008295:lombricine; D001120:Arginine; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022716:taurocyamine kinase; C004946:glycocyamine; D003401:Creatine; D005998:Glycine