Abstract
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 Vmax/Km(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 Vmax/Km(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.
- Copyright © 1979 by Academic Press, Inc.
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