Ethanol unfolds firefly luciferase while competitive inhibitors antagonize unfolding: DSC and FTIR analyses.
Firefly luciferase has gained popularity as a protein model in elucidating anaesthesia mechanism because the bioluminescence of the purified enzyme system is extremely sensitive to volatile anaesthetics. This study analysed the thermal unfolding of firefly luciferase by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR). DSC showed that the transition of firefly luciferase from the folded (N) to unfolded (D) state occurred at 41.7 degrees C with the excess heat flow of 1.6 cal g-1 protein. Ethanol decreased the transition temperature dose dependently. In contrast, luciferin competitors, anilinonaphthalenesulphonate (ANS), toluidinonaphthalenesulphonate (TNS), and myristic acid increased the transition temperature. The competitive inhibitors antagonized unfolding and stabilized the N-state. Ethanol promoted unfolding and stabilized the D-state. Temperature scan by FTIR agreed with the DSC data. The intensities of amide-I' and amide-II' bands started to increase at 20-25 degrees C. This temperature coincides with the temperature where the bioluminescence of firefly luciferase is maximal. The unfolding effect of ethanol was evident even at 5 degrees C. ANS, TNS, and myristic acid completely protected the enzyme from the thermal unfolding. This is the first demonstration that the noncompetitive inhibitors induce the isothermal first-order phase transition in a functional protein, whereas competitive inhibitors protect the enzyme from thermal unfolding. The action mode of competitive inhibitors on firefly luciferase is completely different from that of noncompetitive inhibitors.
Chiou
J S
JS
Department of Anesthesia, DVA Medical Center, Salt Lake City, UT 84148.
Ueda
I
I
D002152:Calorimetry, Differential Scanning; D003460:Crystallization; D000431:Ethanol / Q000737:chemistry*; D008156:Luciferases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000187:drug effects; D008163:Luminescent Measurements; D011487:Protein Conformation / Q000187:drug effects; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D000431:Ethanol; D008156:Luciferases