Abstract
Mutants in which Thr-353 of the Ca(2+)-ATPase of sarcoplasmic reticulum had been replaced with alanine, serine, glutamine, cysteine, valine, aspartate, or tyrosine were analyzed functionally. All the mutations severely affected MgATP binding, whereas ATP binding was close to normal in the alanine, serine, glutamine, and valine mutants. In the serine and valine mutants, the maximum rate of phosphorylation from MgATP was 8- and 600-fold lower, respectively, compared with wild type. Replacement of Mg(2+) with Mn(2+) led to a 1.5-fold enhancement of the maximum phosphorylation rate in the valine mutant and a 5-fold reduction in the wild type. The turnover of the phosphoenzyme formed from MgATP was slowed 1-2 orders of magnitude relative to wild type in the alanine, serine, and valine mutants, but was close to normal in the aspartate and cysteine mutants. Only the serine mutant formed a phosphoenzyme in the backward reaction with P(i), and the hydrolysis of this intermediate was greatly enhanced. Analysis of the functional changes in the mutants in the light of the recent high resolution structure of the Ca(2+)-ATPase crystallized without the MgATP substrate suggests that, in the native activated state of the enzyme, the side chain hydroxyl of Thr-353 participates in important interactions with nucleotide and phosphate, possibly in catalysis, whereas the main chain carbonyl of Thr-353, but not the side chain, may coordinate the catalytic Mg(2+).
Highlights
A fundamental property of the P-type ion transporting ATPases is their ability to bind the substrate MgATP with high affinity and catalyze the phosphorylation of a conserved aspartic acid residue in the presence of activating ions [1]
The formation as well as the further processing of the aspartyl phosphorylated intermediate is associated with protein conformational changes that couple the events in the catalytic site with changes in the ion binding sites leading to ion translocation across the membrane (Fig. 1)
The molecular nature of the binding sites for ATP and the catalytic magnesium ion, as well as the conformational changes involved in energy transduction, are not well understood, and more information is clearly needed about the functions of the individual amino acid residues in the catalytic site
Summary
Ca2ϩ-ATPase, the sarco(endo)plasmic reticulum Ca2ϩ-transporting adenosine triphosphatase (EC 3.6.1.38); lized with bound Ca2ϩ, but without the MgATP substrate [2], has revealed that the cytoplasmic portion of the protein is made up from three distinct domains (A, P, and N) that are rather loosely attached to each other and must move considerably to accomplish substrate binding and energy transduction [2, 3]. Maruyama and co-workers [4] reported that mutation of Thr-353 to serine or alanine resulted in pumps with reduced Ca2ϩ transport activity but with preserved ability to undergo phosphorylation. These observations led to the proposal that the main role of Thr-353 is in the events that take place following phosphorylation, i.e. the conformational changes that are required for release of Ca2ϩ to the lumen or the dephosphorylation [4].
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