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The binding of Thermomyces lanuginosa lipase and its mutants [TLL(S146A), TLL(W89L), TLL(W117F, W221H, W260H)] to the mixed micelles of cis-parinaric acid/ sodium taurodeoxycholate at pH 5.0 led to the quenching of the intrinsic tryptophan fluorescence emission (300–380 nm) and to a simultaneous increase in the cis-parinaric acid fluorescence emission (380–500 nm). These findings were used to characterize the Thermomyces lanuginosa lipase/cisparinaric acid interactions occurring in the presence of sodium taurodeoxycholate | Eur. J. Biochem. 269 1613-1621 2002 FEBS 2002 Binding of Thermomyces Humicola lanuginosa lipase to the mixed micelles of cis-parinaric acid NaTDC Fluorescence resonance energy transfer and crystallographic study Stéohane Yanoudiian1 Maraarita G. Ivanova1 A Marek Brzozowski2 Shamkant A Patkar3. Jesner Vind3. l Bl Bl Allan Svendsen3 and Robert Verger1 1Laboratoire de Lipolyse Enzymatique CNRS-IFR1 Marseille France Structural Biology Laboratory Chemistry Department University of York UK 3Enzyme Research Novozymes A S Bagsvaerd Denmark The binding of Thermomyces lanuginosa lipase and its mutants TLL S146A TLL W89L TlL W117F W221H W260H to the mixed micelles of cis-parinaric acid sodium taurodeoxycholate at pH 5.0 led to the quenching of the intrinsic tryptophan fluorescence emission 300-380 nm and to a simultaneous increase in the cis-parinaric acid fluorescence emission 380-500 nm . These findings were used to characterize the Thermomyces lanuginosa lipase cis-parinaric acid interactions occurring in the presence of sodium taurodeoxycholate.The fluorescence resonance energy transfer and Stern-Volmer quenching constant values obtained were correlated with the accessibility of the tryptophan residues to the cis-parinaric acid and with the lid opening ability of Thermomyces lanuginosa lipase and its mutants . TLL S146A was found to have the highest fluorescence resonance energy transfer. In addition a TLL S146A oleic acid complex was crystallised and its three-dimensional structure was solved. Surprisingly two possible binding modes sn-1 and antisn1 were found to exist between oleic acid and the catalytic cleft of the open conformation of TLL S146A . Both binding modes involved an interaction with tryptophan 89 of the lipase lid in agreement with fluorescence resonance energy transfer experiments.As a consequence we concluded that TLL S146A mutant is not an appropriate substitute for the wild-type Thermomyces lanuginosa lipase for mimicking the interaction between the .
