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 i nhibitors of firefly luciferase in competition with luciferin in mu M range s. They increased the thermal transition temperature. In contrast, 1-alkano ls of the same carbon chain length inhibited the enzyme non-competitively i n mM ranges and decreased the transition temperature. The present study sho wed that the action of fatty acids switched from specific to non-specific w hen the carbon chain length was reduced below C8 (octanoate). The fatty aci ds longer than C10 inhibited the enzyme in mu M ranges whereas those shorte r than C8 required mM ranges to inhibit it. The longer fatty acids increase d whereas shorter fatty acids decreased the transition temperature. The Kil l 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 aci ds is limited. Fatty acids longer than C10 are high-affinity specific binde rs 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 recep tor cavity discriminates specific binders. (C) 1999 Elsevier Science B.V. A ll rights reserved.