title: Effects of high-affinity inhibitors on partial reactions, charge movements, and conformational States of the Ca2+ transport ATPase (sarco-endoplasmic reticulum Ca2+ ATPase).
abstract: The inhibitory effects of thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone, and 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene on the Ca(2+) ATPase were characterized by comparative measurements of sequential reactions of the catalytic and transport cycle, including biochemical measurements and detection of charge movements within a single cycle. In addition, patterns of ATPase proteolytic digestion with proteinase K were derived to follow conformational changes through the cycle or after inhibitor binding. We find that thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone inhibit Ca(2+) binding and catalytic activation as demonstrated with isotopic tracers and lack of charge movement upon addition of Ca(2+) in the absence of ATP. It has been shown previously that binding of these inhibitors requires the E2 conformational state of the ATPase, obtained in the absence of Ca(2+). We demonstrate here that E2 state conformational features are in fact induced by these inhibitors on the ATPase even in the presence of Ca(2+). The resulting dead-end complex interferes with progress of the catalytic and transport cycle. Inhibition by 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene, on the other hand, is related to interference with a conformational transition of the phosphorylated intermediate (E1 approximately P . 2Ca(2+) to E2-P . 2Ca(2+) transition), as demonstrated by increased phosphoenzyme levels and absence of bound Ca(2+) translocation upon addition of ATP. This transition includes large movements of ATPase headpiece domains and transmembrane segments, produced through utilization of ATP-free energy as the "conformational work" of the pump. We conclude that the mechanism of high-affinity Ca(2+) ATPase inhibitors is based on global effects on protein conformation that interfere with ATPase cycling.
authors.affiliation: Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Tadini-Buoninsegni
authors.forename: Gianluca
authors.initials: G
authors.lastname: Bartolommei
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Moncelli
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Tal
authors.forename: David
authors.initials: D
authors.lastname: Lewis
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Inesi
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006873:Hydroquinones / Q000737:chemistry / Q000494:pharmacology; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology; D007550:Isothiuronium / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D055672:Static Electricity; D019284:Thapsigargin / Q000737:chemistry / Q000494:pharmacology
chemical_list: C108416:1,3-dibromo-2,4,6-tris-(methylisothiouronium)benzene; D004791:Enzyme Inhibitors; D006873:Hydroquinones; D007211:Indoles; D007550:Isothiuronium; C019359:2,5-di-tert-butylhydroquinone; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid