Val200 residue in Lys189-Lys205 outermost loop on the A domain of sarcoplasmic reticulum Ca2+-ATPase is critical for rapid processing of phosphoenzyme intermediate after loss of ADP sensitivity

Possible roles of the Lys(189)-Lys(205) outermost loop on the A domain of sarcoplasmic reticulum Ca2+-ATPase were explored by mutagenesis. Both nonconservative and conservative substitutions of Val(200) caused very strong inhibition of Ca2+-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 Ca2+-ATPase at steady state. In contrast, decay of EP formed from ATP in the presence of Ca2+ was strongly inhibited in the mutants. Hydrolysis of E2P formed from P-i in the absence of Ca2+ 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 Ca2+-ATPase, the limitation is likely associated with the Ca2+-releasing step from E2P(.)Ca(2). On the other hand, the rate of activation of dephosphorylated enzyme on high affinity Ca2+ 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 Ca2+-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.