Characterization of Ca2+ ATPase residues involved in substrate and cation binding.
The role of amino acid residues involved in substrate and cation binding was investigated in complementary experiments on Fe(2+)-catalyzed oxidation and cleavage, limited digestion with proteinase K, and mutational analysis. Cleavage at Ser346 was produced by Fe(2+) in the presence of substrate (ATP or AMP-PNP) and Ca(2+), and was attributed to Fe(2+) bound to a Mg(2+) site near Ser346 and neighboring Glu696. Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain was also observed and attributed to Fe(2+) substituting for Mg(2+) in the Mg(2+)-ATP complex bound to the N domain. Mutation of Arg560 or Glu439 within the N domain interfered with nucleotide-dependent ATPase resistance to digestion with proteinase K. Furthermore, mutation of Lys352, Lys684, Thr353, Asp703, or Asp707 within the P domain produced similar interference, consistent with a role of these residues in substrate stabilization at the catalytic site. In a third group of experiments, equilibrium isotherms were obtained with Asn796Ala and Glu309Gln mutants, demonstrating non-cooperative binding of one Ca(2+) per ATPase, as opposed to cooperative binding of two Ca(2+) by WT enzyme. No high-affinity binding by Asp800Asn, Glu771Gln, and Thr799Ala mutants was detected. It was also demonstrated that the conformational transitions involved in enzyme activation and interconversion of Ca(2+) binding and phosphorylation energy, are triggered by Ca(2+) binding to site II and stabilization of Glu309 (M4) and N796 (M6).
Inesi
Giuseppe
G
Department of Biochemistry, University of Maryland, Baltimore 21201, USA. ginesi@umaryland.edu
Ma
Hailun
H
Hua
Suming
S
Toyoshima
Chikashi
C
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D002522:Chlorocebus aethiops; D007501:Iron / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000201:enzymology; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013379:Substrate Specificity; D014162:Transfection
D002413:Cations, Divalent; D007501:Iron; D000252:Calcium-Transporting ATPases; D008274:Magnesium