Cooperativity in the two-domain arginine kinase from the sea anemone Anthopleura japonicus. II. Evidence from site-directed mutagenesis studies

The arginine kinase (AK) from the sea anemone Anthopleura japonicus has an unusual two-domain structure (contiguous dimer; denoted by D1–D2). In a previous report, we suggested cooperativity in the contiguous dimer, which may be a result of domain–domain interactions, using MBP-fused enzymes. To further understand this observation, we inserted six-Lys residues into the linker region of the two-domain AK (D1–K6–D2 mutant) using His-tagged enzyme. The dissociation constants, Ka and Kia, of the mutant were similar to those of the wild-type enzyme but the catalytic constant, kcat, was decreased to 28% that of the wild-type, indicating that some of the domain–domain interactions are lost due to the six-Lys insertion. Y68 plays a major role in arginine binding in the catalytic pocket in Limulus AK, and introduction of mutation at the Y68 position virtually abolishes catalytic activity. Thus, the constructed D1(Y68G)–D2 and D1–D2(Y68G) mutants mimic the D1(inactive)–D2(active) and D1(active)–D2(inactive) enzymes, respectively. The kcat values of both Y68 mutants were decreased to 13–18% that of the wild-type enzyme, which is much less than the 50% level of the two-domain enzyme. Thus, it is clear that substrate-binding to both domains is necessary for full expression of activity. In other words, substrate-binding appears to act as the trigger of the functional cooperativity in two-domain AK.