Specific dicyclohexylcarbodiimide inhibition of the E-P + H2O equilibrium E + Pi reaction and ATP equilibrium Pi exchange in sarcoplasmic reticulum adenosinetriphosphatase.
Treatment of sarcoplasmic reticulum adenosinetriphosphatase (ATPase) with N,N'-dicyclohexylcarbodiimide is known to produce total inhibition of calcium binding and enzyme activity. However, we now find that treatment with lower reagent:protein ratios produces selective inhibition of hydrolytic Pi cleavage, enzyme phosphorylation with Pi, and ATP in equilibrium Pi exchange, while calcium binding and enzyme phosphorylation with ATP remain largely unaffected. This specific inhibition is attributed to derivatization of residues which are normally involved in acid-base-assisted catalysis of the hydrolytic reaction and its reversal, but are not involved in calcium binding or in the mechanism of phosphoryl transfer from ATP to the enzyme. This specific inhibition is prevented by the presence of micromolar calcium during the incubation with the inhibitor, evidently through an allosteric effect of calcium binding on the catalytic site. We also find that the initial adducts formed between ATPase residues and N,N'-dicyclo[14C]carbodiimide undergo further degradation with release of radioactive product into the medium, while the protein residues remain inactivated probably by linkage with neighboring residues. Therefore, the stoichiometry of radioactive labeling underestimates the actual number of inactivated residues.
Scofano
H M
HM
Barrabin
H
H
Lewis
D
D
Inesi
G
G
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002122:Calcium Chloride / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002234:Carbodiimides / Q000494:pharmacology*; D002250:Carbon Radioisotopes; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D002234:Carbodiimides; D002250:Carbon Radioisotopes; D010710:Phosphates; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D002122:Calcium Chloride