Ueda I, Suzuki A
Anesthesia 112A, Department of Veterans Administration Medical Center, and University of Utah School of Medicine, Salt Lake City, Utah 84148 USA.
Biophys J. 1998 Aug;75(2):1052-7. doi: 10.1016/S0006-3495(98)77594-0.
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.
萤火虫荧光素酶在氧气存在下与三磷酸腺苷（ATP）和荧光素（L）混合时会发出一阵光。本研究比较了长链正构醇（1 - 癸醇至1 - 十八醇）和脂肪酸（癸酸至十八烷酸）对萤火虫荧光素酶的影响。脂肪酸对萤火虫荧光素酶的抑制作用比正构醇更强。肉豆蔻醇在13.6微摩尔时使光强度降低50%（半数抑制浓度，IC50），而肉豆蔻酸的IC50为0.68微摩尔。根据迈耶 - 奥弗顿规则，脂肪酸作为抑制剂的效力比相应的醇大约强12000倍。双倒数作图（Lineweaver - Burk plot）表明，肉豆蔻酸与荧光素竞争抑制萤火虫荧光素酶，而肉豆蔻醇则是非竞争性抑制。差示扫描量热法（DSC）显示，在约39℃发生不可逆的热转变，转变焓变（ΔHcal）为1.57卡/克。通过不可逆变化完成一半时的温度来评估配体对转变的影响。醇类降低而脂肪酸升高萤火虫荧光素酶的热转变温度。科什兰德的过渡态理论（《科学》，1963年，第142卷，第1533 - 1541页）指出，与底物识别位点结合的配体会诱导酶处于过渡态，该状态比天然状态更稳定，能抵抗热扰动。长链脂肪酸与荧光素识别位点结合并在过渡态稳定蛋白质构象，从而抵抗热变性。艾林的解折叠理论（《科学》，1966年，第154卷，第1609 - 1613页）假定麻醉剂和醇类非特异性地结合到蛋白质的界面区域并可逆地使构象解折叠。目前的结果表明，醇类不与荧光素竞争，而是通过使蛋白质解折叠非特异性地抑制萤火虫荧光素酶。脂肪酸是受体结合剂，并在过渡态稳定蛋白质构象。
