Cytochrome c oxidase inhibition by anesthetics: thermodynamic analysis.

Abstract

The thermodynamic parameters that characterize the inhibition of cytochrome c oxidase activity, in rat liver submitochondrial particles, by n-butanol, tetracaine, and dibucaine were obtained. Three equilibria were assumed in order to account for the data: for the interaction of inhibitor with the native state of the enzyme, for the interaction of inhibitor with the thermally (reversibly) denatured state, and for the change between the native and thermally denatured states. Inhibition results from interaction with both the native and denatured states but, because the interaction is stronger with the denatured than with the native state, the native/denatured equilibrium is shifted to the right by the anesthetics. The enthalpies ofinteraction are -2.3, -4.7, and 3.7 kcal/mol (1 cal = 4.18 J) for the native state and -10, -6, and -14 kcal/mol for the denatured state, for n-butanol, tetracaine, and dibucaine, respectively. These values are much smaller than the previous estimates obtained by using the assumption that anesthetics interact only with the thermally denatured state of enzymes (e.g., -81 kcal/mol for tetracaine inhibition of luciferase). Our results suggest that local anesthetics inhibit enzyme activity by causing a reversible perturbation of protein conformation. The magnitude of the perturbation is much smaller (in energetic terms) than that which accompanies thermal denaturation.Johnson et al. (1, 2) showed how it is possible to obtain thermodynamic information on inhibitor binding to proteins by studying the temperature dependence of enzyme activity. The information thus obtained can reveal whether the inhibitor binds preferentially to the native or denatured forms of the enzyme, and the magnitude of the enthalpy and entropy changes may indicate whether any appreciable conformational alterations accompany the binding. carried out thermodynamic analyses of the inhibition of bacterial luminescence and respiration by anesthetics and other substances. More recently, the same approach was used by Ueda et al. (5, 6) to study the inhibition ofpurified firefly luciferase by general and local anesthetics. In each of these studies, large negative enthalpy values were found for the binding ofanesthetics to the denatured form ofthe enzyme [e. g., -81 kcal/mol for luciferase inhibition by tetracaine (6)]. These values were of the same order of magnitude as, but of opposite sign to, the enthalpies of denaturation reported for the same systems. On this basis, Eyring et al. (7) proposed that anesthetics cause a major unfolding of protein structure, comparable in extent to protein denaturation. The well known pressure reversal of anesthesia (2, 8) was also explained in terms of this unfolding: if a volume increase accompanies the unfolding, pressure should counteract the anesthetic effect.There has been some confusion in the literature over the sign of the enthalpy change accompanying anesthetic binding, with the same value being given as positive or negative in different places. The correct sign for AH3 in t...

Influence of detergent polar and apolar structure upon the temperature dependence of beef heart cytochrome c oxidase activity

“…The temperature dependence of cytochrome c oxidase catalyzed electron transport in each detergent that we tested agrees quite well with the previous studies that have been performed in phospholipid, detergent, or membrane environments, e.g., Smith & Newton (1968), Raison et al (1971) (1980). The largest apparent discrepancy between the present data and those that had been obtained earlier is the absence of a clear break in any of the Arrhenius plots of our data between 20 and 25 °C (refer to Figures 3 and 4) although some previous data also lacked a clear break (Denes & Stanacev, 1979;Yohsida et al, 1979;Vanderkooi & Chazotte, 1982). In each of our detergent systems, some curvature in the Arrhenius plot was evident, and two lines could have been drawn through the data that intersected between 20 and 25 °C.…”

Structural perturbation of the a3-CuB site in mitochondrial cytochrome c oxidase by alcohol solvents

“…They have shown that several enzymes, including cytochrome c oxidase, are inhibited by these local anesthetics. In analyzing thermodynamic parameters that characterize the interaction of anesthetics with cytochrome oxidase, they suggested that inhibition of enzyme activity was caused by a reversible perturbation of protein comformation, to a degree much smaller than that required for thermal denaturation (Vanderkooi & Chazotte, 1982). This suggests that an ethanol-induced increase in protein fluidity, in addition to specific binding near the metal sites in cytochrome oxidase, may cause changes that we observe in the -form of the enzyme.…”

The inhibition of a membrane-bound enzyme as a model for anaesthetic action and drug toxicity

“…Quinacrine (Casanovas et al, 1985a) was also observed to bind to cytochrome c oxidase. A variety of alcohols (butan-I -ol, pentan-I -ol, hexan-1 -ol and benzyl alcohol) as well as several amine local anaesthetics were observed to inhibit at multiple sites along the mitochondrial electron-transport chain, including the cytochrome c oxidase site (Chazotte & Vanderkooi, 1981;Vanderkooi & Chazotte, 1982). Six fully reduced flavin analogues have also been observed to bind to cytochrome c oxidase, with dissociation constants ranging from 5 to 8 ,UM (Ahmad et al, 1982).…”

“…Cytochrome c oxidase has been previously used as a model for drug-membrane enzyme interactions (Singer, 1980(Singer, , 1982Chazotte & Vanderkooi, 1981;Vanderkooi & Chazotte, 1982;Casanovas et al, 1983Casanovas et al, , 1985a. A variety of amine local anaesthetics (11 in all) with a significant range of lipophilicities (as measured by their octanol/water partition coefficients) demonstrated both competitive and mixed-type inhibition.…”