Molecular Pharmacology, Vol 16, 189-195, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

Role of Phospholipid (Cardiolipin) in the Modulation of Substrate and Inhibitor Interactions with Erythrocyte Acetylcholinesterase

¹ Laboratory of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, B.C., V6T 1W5 Canada

The hydrolysis of acetylcholine by bovine erythrocyte acetylcholinesterase shows a break in the Arrhenius plot around 20°. Evidence is presented that the cardiolipin-associated enzyme is modulated by a temperature dependent conformational rearrangement of the active site induced by the binding of substrate and inhibitor to the enzyme. Neither carbamylation by o-nitrophenoldimethylcarbamate, decarbamylation nor the maximum velocity of acetylcholine hydrolysis (rate limited by deacetylation) showed a non-linear Arrhenius plot. The break the Arrhenius plot of acetylcholine hydrolysis appeared to parallel the nonlinear temperature dependence of V_max/K_m(app) which reflects the conformational rearrangement of the enzyme-substrate complex, the rate limiting step at subsaturating substrate concentrations. Treatment of the enzyme with high salt and phosphate, which abolishes the non-linear Arrhenius plot and permits the extraction of cardiolipin from the enzyme by chloroform/methanol, gave linear plots for the temperature dependence of V_max/K_m(app), indicating that in these conditions the rate-limiting step was altered or no longer modulated by temperature.

Above the transition temperature fluoride inhibited the cardiolipin-associated enzyme with a Hill slope greater than 1.0 (-n = 1.4), whereas below the transition temperature or in high salt and phosphate treated cardiolipin-dissociated enzyme the corresponding negative Hill slope was 1.0. The rate of irreversible inhibition at the active site by N,N'-dicyclohexylcarbodiimide also exhibited a break at around 20° , the large increase in the entropy of the reaction above this temperature being consistent with a conformational rearrangement of the active site.