Reactivity of sarcoplasmic reticulum adenosinetriphosphatase with iodoacetamide spin-label: evidence for two conformational states of the substrate binding sites.
The labeling kinetics of sarcoplasmic reticulum ATPase with the iodoacetamide spin probe N-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide were followed under conditions designed to selectively label all reactive groups. Approximately 1 mol of spin-label reacted per one 100 000-dalton ATPase chain, indicating only one residue on the enzyme had been labeled. One uniform rate of labeling was observed in the presence of Ca2+. When substrate was then added, approximately one-half of the residues showed a 10-fold increase in labeling rate while the remaining residues reacted at the initial, slower rate. Sequential labeling experiments further established that the two labeling rates correspond to the coexistence of two conformational state of the enzyme. Both Ca2+ and substrate are required to obtain an equal distribution between states, and the effect is completely reversed when substrate is removed. The iodoacetamide spin probe is known to be highly sensitive to the conformation of the ATPase binding pocket, and the residue labeled here is the one which generates broadening in the electron paramagnetic resonance spectrum on substrate binding. Due to the unique selectively of the labeling reaction, it is suggested that when both substrate and Ca2+ are bound to the enzyme, conditions which are precursory to enzyme phosphorylation, two specific conformations of the binding pocket exist in approximately at 50:50 ratio.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002122:Calcium Chloride / Q000494:pharmacology; D003497:Cyclic N-Oxides / Q000494:pharmacology*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013113:Spin Labels / Q000494:pharmacology*
D003497:Cyclic N-Oxides; D013113:Spin Labels; D000266:Adenylyl Imidodiphosphate; C003028:N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide; D000251:Adenosine Triphosphatases; D002122:Calcium Chloride