Possible roles of the Lys(189)-Lys(205) outermost loop on the A domain of sarcoplasmic reticulum Ca(2+)-ATPase were explored by mutagenesis. Both nonconservative and conservative substitutions of Val(200) caused very strong inhibition of Ca(2+)-ATPase activity, whereas substitutions of other residues on this loop reduced activity only moderately. All of the Val(200) mutants formed phosphoenzyme intermediate (EP) from ATP. Isomerization from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was not inhibited in the mutants, and a substantially larger amount of E2P actually accumulated in the mutants than in wild-type sarcoplasmic reticulum Ca(2+)-ATPase at steady state. In contrast, decay of EP formed from ATP in the presence of Ca(2+) was strongly inhibited in the mutants. Hydrolysis of E2P formed from P(i) in the absence of Ca(2+) was also strongly inhibited but was faster than the decay of EP formed from ATP, indicating that the main kinetic limitation of the decay comes after loss of ADP sensitivity but before E2P hydrolysis. On the basis of the well accepted mechanism of the Ca(2+)-ATPase, the limitation is likely associated with the Ca(2+)-releasing step from E2P.Ca(2). On the other hand, the rate of activation of dephosphorylated enzyme on high affinity Ca(2+) binding was not altered by the substitutions. In light of the crystal structures, the present results strongly suggest that Val(200) confers appropriate interactions of the Lys(189)-Lys(205) loop with the P domain in the Ca(2+)-released form of E2P. Results further suggest that these interactions, however, do not contribute much to domain organization in the dephosphorylated enzyme and thus would be mostly lost on E2P hydrolysis.