Specific and non-specific binding of long-chain fatty acids to firefly luciferase: cutoff at octanoate.
Firefly luciferase emits a burst of light when the substrates luciferin and ATP are mixed in the presence of oxygen. We (I. Ueda, A. Suzuki, Biophys. J. 75 (1998) 1052-1057) reported that long-chain fatty acids are specific inhibitors of firefly luciferase in competition with luciferin in microM ranges. They increased the thermal transition temperature. In contrast, 1-alkanols of the same carbon chain length inhibited the enzyme non-competitively in mM ranges and decreased the transition temperature. The present study showed that the action of fatty acids switched from specific to non-specific when the carbon chain length was reduced below C8 (octanoate). The fatty acids longer than C10 inhibited the enzyme in microM ranges whereas those shorter than C8 required mM ranges to inhibit it. The longer fatty acids increased whereas shorter fatty acids decreased the transition temperature. The Hill coefficients of longer chain bindings were less than one whereas those of shorter chain were more than one. The shorter fatty acids interacted with the enzyme cooperatively at multiple sites. Binding of the longer fatty acids is limited. Fatty acids longer than C10 are high-affinity specific binders and followed Koshland's induced-fit model. Those shorter than C8 are low-affinity non-specific denaturants and followed Eyring's rate process model. These results contradict the general consensus that the size of the receptor cavity discriminates specific binders.
Matsuki
H
H
Department of Anesthesia, Anesthesia Service 112A, DVA Medical Center, and University of Utah School of Medicine, 500 Foothill Blvd. , Salt Lake City, UT 84148, USA.
Suzuki
A
A
Kamaya
H
H
Ueda
I
I
D000818:Animals; D020148:Butyric Acid / Q000494:pharmacology*; D002208:Caproates / Q000494:pharmacology*; D002210:Caprylates / Q000494:pharmacology*; D001517:Coleoptera; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008156:Luciferases / Q000037:antagonists & inhibitors*; D008163:Luminescent Measurements; D011489:Protein Denaturation; D012995:Solubility; D013696:Temperature
D002208:Caproates; D002210:Caprylates; D004791:Enzyme Inhibitors; D020148:Butyric Acid; C037652:hexanoic acid; D008156:Luciferases; C031492:octanoic acid