Stichopus japonicus arginine kinase: gene structure and unique substrate recognition system

Arginine kinase from the sea cucumber Stichopus japonicus underwent a uniqu e molecular evolution. Unlike the monomeric 40 kDa arginine kinases from mo lluscs and arthropods, Stichopus arginine kinase is dimeric, the same as cy toplasmic isoenzymes of the vertebrate creatine kinases. Its entire amino a cid sequence is more similar to creatine kinases than to other arginine kin ases, but the guanidino specificity region (GS region) is of the arginine k inase type. To elucidate its unusual evolution, the structure of the Sticho pus arginine kinase gene was determined. It consisted of seven exons and si x introns, and a part of the exon 2 of the Stichopus gene corresponds to th e GS region. Compared with the structure of the human muscle creatine kinas e gene (seven exons, six introns), the splice junctions of five introns wer e conserved exactly between the two genes, suggesting that these introns ha d been conserved for at least 500 million years. The entire sequence of Sti chopus arginine kinase is distinctly included in the creatine kinase cluste r in all tree construction methods examined. On the other hand, if the tree is constructed only from sequences corresponding to Stichopus exon 2, it i s placed in the arginine kinase cluster. Thus we conclude that Stichopus ar ginine kinase evolved not from the arginine kinase gene but from the creati ne kinase gene, and suggest that its GS region, determining substrate speci ficity, has been replaced by an arginine kinase type via exon shuffling. In typical arginine kinases four residues, Ser(63), Gly(64), Val(65) and Tyr( 68) (numbering from the Limulus polyphemus sequence), in the GS region are highly conserved and are associated with substrate binding. Among them, Tyr (68) appears to play a crucial role by forming a hydrogen bond with the sub strate, and is conserved exactly in all arginine kinases. However, in Stich opus arginine kinase, none of these four conserved residues were present. N evertheless, the enzyme displays an affinity for the substrate arginine (K- m = 0.8 mM) comparable with other arginine kinases. This implies that a com pletely different substrate-binding system has been developed in Stichopus arginine kinase. We propose that the His(64) in Stichopus arginine kinase a cts as a substitute for the Tyr(68) in other arginine kinases, and that the imidazole ring of His(64) is hydrogen bonded with the substrate arginine, thus stabilizing it.