Fifty-four homolog (Ffh) and FtsY are the central components of the signal recognition particle secretory pathway of bacteria. In this study, the core domain and active sites of FtsY and Ffh from Streptomyces coelicolor, which are responsible for guanosine triphosphate (GTP) hydrolysis, were identified using site-directed mutagenesis. Mutations were introduced to the conserved GXXGXGK loop of the putative GTP binding site. Mutation of the Lys residue to Gly in both FtsY and Ffh NG domains significantly decreased the GTPase activity and GTP binding affinity. Furthermore, a structural model of the ternary complex of FtsY/Ffh NG domains and the non-hydrolyzable GTP analog guanylyl 5'-(beta,gamma-methylenediphosphonate) also revealed that each Lys residue in GXXGXGK of FtsY and Ffh provides the predicted hydrogen bond required for GTP binding. However, in FtsY not in Ffh, mutation of the first Gly residue in the GXXGXGK loop disrupted the GTPase activity. In addition, protease-digesting test demonstrated that NG protein with the mutation of Lys residue was decomposed more easily. Western blot analysis suggested that in Streptomyces coelicolor, FtsY is present in the membrane fraction and Ffh in the cytosol fraction during the mid-log phase of growth. These results indicated that Lys residue in the putative GTP binding loop was the crucial residue for the GTPase activity of NG domain.