Irreversible phase transition of firefly luciferase: contrasting effects of volatile anesthetics and myristic acid.
Firefly luciferase (FFL) has been used as a lipid-free protein model to study direct interaction of anesthetics with proteins. FFL emits a burst of light when luciferin and ATP are added in the presence of oxygen. Volatile anesthetics inhibited FFL at mM ranges, while myristic acid inhibited it at microM range. Despite the large difference, octanol/water partition coefficients of both myristic acid and halothane are 199. Differential scanning calorimetry (DSC) showed that thermal transition occurred at 38.5 degreesC with excess enthalpy of denaturation of 91.9 kcal mol-1. The transition, however, was irreversible. According to the irreversible transition kinetics, the anesthetic effects were evaluated by the temperature where the irreversible transition is half completed (T1/2). Volatile anesthetics decreased T1/2 at mM ranges, while myristic acid and oxyluciferyladenylate (luciferin competitor) increased it at microM ranges. Luciferin is a heterocyclic carboxylate and acylates AMP. Carboxyl group of myristic acid appears to make a high affinity contact to the luciferin-recognition sites. The induced-fit theory states that binding of substrates induces the enzyme into high-energy transition state. Myristic acid stabilized FFL at the transition state, which resisted thermal denaturation. Anesthetics destabilized FFL by reversibly unfolding the protein into less active intermediate states and promoted irreversible transition when the temperature is elevated.
Ueda
I
I
Anesthesia Service, Department of Veterans Affairs Medical Center, Salt Lake City, UT 84148, USA. iueda3032@pol.net
Suzuki
A
A
D018685:Anesthetics, Inhalation / Q000494:pharmacology*; D000818:Animals; D002152:Calorimetry, Differential Scanning; D001517:Coleoptera; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008163:Luminescent Measurements; D019814:Myristic Acid / Q000494:pharmacology*; D017510:Protein Folding; D013379:Substrate Specificity; D013696:Temperature
D018685:Anesthetics, Inhalation; D004791:Enzyme Inhibitors; D019814:Myristic Acid; D008156:Luciferases