Kinetic and Thermodynamic Aspects of the Mechanism of General Anesthesia in a Model System of Firefly Luminescence in Vitro

Cell-free firefly-tail extract emits light when ATP is added. The flash intensity follows first-order kinetics. Methoxyflurane, chloroform, halothane, enflurane, and fluroxene inhibited this reaction. Tensions which inhibited flash intensity 50 per cent were 2.1 X 10-3 atm, 9.0 X 10-4 atm, 1.04 X 10-2 atm, 2.0 X 10-2 atm, and 5.1 X 10-2 atm, respectively. These values correlate better with oil/gas partition coefficients than with hydrate-dissociation pressures. Thermodynamic analysis showed that the inhibition mechanism of anestheties is identical to reversible thermal inactivation of the enzyme. In this reaction the origital “folded” enzyme became inactive by transformation into the “unfolded” or expanded type. Inhibition was accompanied by a high heat of reaction (ΔH: −80,000 to −89,900 cal/mol) and a large entropy change (ΔS: −277 to −320 entropy units), and was non-competitive with ATP binding. The magnitudes of the heat of reaction and the entropy change support the theory that anesthetics act at a hydrophobic site of luciferase, inducing a structural change of the enzyme to form the “unfolded” type. The correlation between the ED20's of the anesthetics and their oil/gas partition coefficients together with the enzyme kinetic data indicates that the site of anesthetic action may be hydrophobic.