Arginine kinase (AK) from the clam Corbicula japonica is a unique enzyme in that it has an unusual two-domain structure with molecular mass of 80 kDa. It lacks two functionally important amino acid residues, Asp-62 and Arg-193, which are conserved in other 40 kDa AKs and are assumed to be key residues for stabilizing the substrate-bound structure. K m arg and Vmax values for the recombinant two-domain AK were determined. These values were close to those of usual 40 kDa AKs, although Corbicula AK lacks the functionally important Asp-62 and Arg-193. Domain 2 of Corbicula AK was separated from the two-domain enzyme and was expressed in Escherichia coli. Domain 2 still exhibited activity. However, kinetic parameters for domain 2 appeared to be slightly, but significantly, different from those of two-domain AK. Thus, it is likely that the formation of the contiguous dimer alters the kinetic properties of its constituent domains significantly. Comparison of K d arg and K m arg for two-domain AK and its domain 2 showed that the affinity of the enzyme for arginine is greater in the presence of substrate ATP than in its absence. Presumably this difference is correlated with the large structural differences in the enzyme in the presence or absence of substrate, namely open and closed structures. We expressed three mutants of Corbicula AK domain 2 (His-60 to Gly or Arg, Asp-197 to Gly), and determined their K m arg and Vmax values. The affinity for the substrate arginine in mutant enzymes was reduced considerably, accompanied by a decrease in Vmax. These results suggest that His-60 and Asp-197 affect the substrate binding system, and are consistent with the hypothesis that a hydrogen bond is formed between His-60 and Asp-197 in Corbicula AK as a substitute for the Asp-62 and Arg-193 bond in normal AKs.