Glutamate-183 in the conserved TGES motif of domain A of sarcoplasmic reticulum Ca2+-ATPase assists in catalysis of E2/E2P partial reactions

The recently determined crystal structures of the sarcoplasmic reticulum Ca2+-ATPase show that in the E1Ca2 form, domain A is almost isolated from the other cytoplasmic domains, P and N, whereas in E-2, domain A has approached domains P and N, with E183 of the highly conserved P-type ATPase signature sequence TGES in domain A now being close to the phosphorylated aspartate in domain P, thus raising the question whether E183 acquires a catalytic role in E-2 and E2P conformations. This study compares the partial reactions of mutant E183A and wild-type Ca2+-ATPase, using transient and steady-state kinetic measurements. It is demonstrated that dephosphorylation of the E2P phosphoenzyme intermediate, as well as reverse phosphorylation of E-2 with P-ir is severely inhibited in the mutant. Furthermore, the apparent affinity of E-2 for the phosphoryl transition state analog vanadate is reduced by three orders of magnitude, consistent with a destabilization of the transition state complex, and the mutant displays reduced apparent affinity for P-i in the E-2 form. The E1Ca2 conformation, on the other hand, shows normal phosphorylation with ATP and normal Ca2+ binding properties, and the rates of the conformational transitions E1PCa2 --> E2P and E-2 - E1Ca2 are only 2- to 3-fold reduced, relative to wild type. These results, which likely can be generalized to other P-type ATPases, indicate that E183 is critical for the phosphatase function of E-2 and E2P, possibly interacting with the phosphoryl group or attacking water in the transition state complex, but is of little functional importance in E-1 and E1P.