Firefly luciferase emits a burst of light when mixed with ATP and luciferin (L) in the presence of oxygen. This study compared the effects of long-chain n-alcohols (1-decanol to 1-octadecanol) and fatty acids (decanoic to octadecanoic acids) on firefly luciferase. Fatty acids were stronger inhibitors of firefly luciferase than n-alcohols. Myristyl alcohol inhibited the light intensity by 50% (IC50) at 13.6 microM, whereas the IC50 of myristic acid was 0.68 microM. According to the Meyer-Overton rule, fatty acids are approximately 12,000-fold stronger inhibitors than corresponding alcohols. The Lineweaver-Burk plot showed that myristic acid inhibited firefly luciferase in competition with luciferin, whereas myristyl alcohol inhibited it noncompetitively. The differential scanning calorimetry (DSC) showed that an irreversible thermal transition occurred at approximately 39 degrees C with a transition DeltaHcal of 1.57 cal g-1. The ligand effects on the transition were evaluated by the temperature where the irreversible change is half completed. Alcohols decreased whereas fatty acids increased the thermal transition temperature of firefly luciferase. Koshland's transition-state theory (Science. 1963. 142:1533-1541) states that ligands that bind to the substrate-recognition sites induce the enzyme at a transition state, which is more stabilized than the native state against thermal perturbation. The long-chain fatty acids bound to the luciferin recognition site and stabilized the protein conformation at the transition state, which resisted thermal denaturation. Eyring's unfolding theory (Science. 1966. 154:1609-1613) postulates that anesthetics and alcohols bind nonspecifically to interfacial areas of proteins and reversibly unfold the conformation. The present results showed that alcohols do not compete with luciferin and inhibit firefly luciferase nonspecifically by unfolding the protein. Fatty acids are receptor binders and stabilize the protein conformation at the transition state.