Importance of Thr-353 of the conserved phosphorylation loop of the sarcoplasmic reticulum Ca2+-ATPase in MgATP binding and catalytic activity.
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+).
Clausen
J D
JD
Department of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark.
McIntosh
D B
DB
Woolley
D G
DG
Andersen
J P
JP
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013912:Threonine / Q000378:metabolism*
D013912:Threonine; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases