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

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National Academy of Sciences

RESUMO

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 E2, 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 E2 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 E2 with Pi, is severely inhibited in the mutant. Furthermore, the apparent affinity of E2 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 Pi in the E2 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 E2 → 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 E2 and E2P, possibly interacting with the phosphoryl group or attacking water in the transition state complex, but is of little functional importance in E1 and E1P.

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