Stichopus arginine kinase (AK) is a unique enzyme in that it evolved not from the AK gene but from the creatine kinase (CK) gene: the entire amino acid sequence is homologous with other CKs apart from the guanidine specificity region (GS region), which is identical in structure to that of AK. Ten independent mutations were introduced around the GS region in Stichopus AK. When an insertion or deletion was introduced near the GS region, the Vmax of the mutant enzyme was dramatically decreased to less than 0.1% of the wild type, suggesting that the length of the GS region is crucial for the recognition of the guanidine substrate. Replacement of Phe63 and Leu65 to Gly in the Stichopus enzyme caused a remarkable increase in the Kmarg. This indicates that Phe63 and Leu65 are associated with the arginine substrate-binding affinity. The hydrogen bond formed between the Asp62 and Arg193 residues is thought to play a key role in stabilizing the closed substrate-bound structure of AK. Mutants that eliminated this hydrogen bond had a considerably decreased Vmax, accompanied by a threefold increase in Kmarg. It is noted that the value of the Kmarg of the mutants became very close to the Kdarg value of the wild type. Six independent mutations were introduced in the GS region of Danio M-CK. Almost equivalent values of Kmcr and Kdcr in all of the mutants indicated that a typical synergism was completely lost. The results suggested that the Ile69 to Gly mutant, displaying a high Kmcr and a low Vmax, plays an important role in creatine-binding. This is consistent with the observation that in the structure of Torpedo CK, Ile69 provides a hydrophobic pocket to optimize creatine-binding.