The Conformational Changes of the Fungal Lipase from Humicola lanuginosa Induced by the Formation of Bile Salt and Mixed Micelles

Abstract

The effect of sodium taurodeoxycholate and a mixture of sodium taurodeoxycholate/phosphatidyl-choline on the activation of fungal lipase from Humicola lanuginosa (HLL) was investigated by monitoring the specific activity and the changes in intrinsic protein fluorescence. A large increase in the inactivation rate was observed at about the critical micellar concentration of bile salt. On the contrary, a high activation of lipase was achieved by the presence of mixed micelles. Steady-state fluorescence quenching measurements were performed to resolve the fluorescence contribution of W89 residue in emission of four-tryptophan-containing HLL lipase. The W89 residue is located in the “lid” helix which participates in interfacial activation of the enzyme. The assignment of W89 residue was confirmed by use of the W89F mutant and inhibited form of lipase. The FQRS (fluorescence quenching resolved spectra) method was used to decompose the total emission spectrum of HLL lipase. The FQRS results show that the fluorescence of the W89 residue is similar in inhibited and inactivated HLL lipase and exhibits a maximum of emission at about 345 ± 1 nm (λex = 295 nm). In the mixed micelle solution the fluorescence of the W89 residue may be resolved into two components, with fluorescence maxima at 337 and 347 nm, respectively (λex = 295 nm). It is concluded that HLL lipase undergoes a conformational transition upon specific interactions with both anionic and mixed micelles, resulting in a change in the microenvironment of the W89 residue.