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

Abstract

The recently determined crystal structures of the sarcoplasmic reticulum Ca²⁺-ATPase show that in the E ₁Ca₂ 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 E ₂P 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 E ₂P phosphoenzyme intermediate, as well as reverse phosphorylation of E ₂ with P_i, 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 E ₁Ca₂ conformation, on the other hand, shows normal phosphorylation with ATP and normal Ca²⁺ binding properties, and the rates of the conformational transitions E ₁PCa₂ → E ₂P and E ₂ → E ₁Ca₂ 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 E ₂P, possibly interacting with the phosphoryl group or attacking water in the transition state complex, but is of little functional importance in E ₁ and E ₁P.