title: Asparagine 706 and glutamate 183 at the catalytic site of sarcoplasmic reticulum Ca2+-ATPase play critical but distinct roles in E2 states.
abstract: Mutants with alteration to Asn(706) of the highly conserved (701)TGDGVND(707) motif in domain P of sarcoplasmic reticulum Ca(2+)-ATPase were analyzed for changes in transport cycle kinetics and binding of the inhibitors vanadate, BeF, AlF, and MgF. The fluorides likely mimic the phosphoryl group/P(i) in the respective ground, transition, and product states of phosphoenzyme hydrolysis (Danko, S., Yamasaki, K., Daiho, T., and Suzuki, H. (2004) J. Biol. Chem. 279, 14991-14998). Binding of BeF, AlF, and MgF was also studied for mutant Glu(183) --> Ala, where the glutamate of the (181)TGES(184) motif in domain A is replaced. Mutations of Asn(706) and Glu(183) have in common that they dramatically impede the function of the enzyme in E2 states, but have little effect in E1. Contrary to the Glu(183) mutant, in which E2P slowly accumulates (Clausen, J. D., Vilsen, B., McIntosh, D. B., Einholm, A. P., and Andersen, J. P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2776-2781), E2P formation was not detectable with the Asn(706) mutants. Differential sensitivities of the mutants to inhibition by AlF, MgF, and BeF made it possible to distinguish different roles of Asn(706) and Glu(183). Hence, Asn(706) is less important than Glu(183) for gaining the transition state during E2P hydrolysis but plays critical roles in stabilization of E2P ground and E2.P(i) product states and in the major conformational changes associated with the Ca(2)E1P --> E2P and E2 --> Ca(2)E1 transitions, which seem to be facilitated by interaction of Asn(706) with domain A.
authors.affiliation: Department of Physiology, Institute of Physiology and Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark.
authors.forename: Johannes D
authors.initials: JD
authors.lastname: Clausen
authors.forename: David B
authors.initials: DB
authors.lastname: McIntosh
authors.forename: David G
authors.initials: DG
authors.lastname: Woolley
authors.forename: Anne Nyholm
authors.initials: AN
authors.lastname: Anthonisen
authors.forename: Bente
authors.initials: B
authors.lastname: Vilsen
authors.forename: Jens Peter
authors.initials: JP
authors.lastname: Andersen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001216:Asparagine; D001665:Binding Sites; D002118:Calcium / Q000493:pharmacokinetics; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D005459:Fluorides / Q000737:chemistry; D018698:Glutamic Acid; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010710:Phosphates; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D014638:Vanadates / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D010710:Phosphates; D018698:Glutamic Acid; D014638:Vanadates; D001216:Asparagine; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D005459:Fluorides; D002118:Calcium