Glutamate-183 in the conserved TGES motif of domain A of sarcoplasmic reticulum Ca super(2+)-ATPase assists in catalysis of E sub(2)/E sub(2)P partial reactions

The recently determined crystal structures of the sarcoplasmic reticulum Ca super(2+)-ATPase show that in the E sub(1)Ca sub(2) form, domain A is almost isolated from the other cytoplasmic domains, P and N, whereas in E sub(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 sub(2) and E sub(2)P conformations. This study compares the partial reactions of mutant E183A and wild-type Ca super(2+)-ATPase, using transient and steady-state kinetic measurements. It is demonstrated that dephosphorylation of the E sub(2)P phosphoenzyme intermediate, as well as reverse phosphorylation of E sub(2) with P sub(i), is severely inhibited in the mutant. Furthermore, the apparent affinity of E sub(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 sub(i) in the E sub(2) form. The E sub(1)Ca sub(2) conformation, on the other hand, shows normal phosphorylation with ATP and normal Ca super(2+) binding properties, and the rates of the conformational transitions E sub(1)PCa sub(2) -> E sub(2)P and E sub(2) -> E sub(1)Ca sub(2) 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 sub(2) and E sub(2)P, possibly interacting with the phosphoryl group or attacking water in the transition state complex, but is of little functional importance in E sub(1) and E sub(1)P.