Binding of Thermomyces (Humicola) lanuginosa lipase to the mixed micelles of cis-parinaric acid/NaTDC.
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 wild-type enzyme and lipids.
Yapoudjian
Stéphane
S
Laboratoire de Lipolyse Enzymatique CNRS-IFR1, Marseille, France.
Ivanova
Margarita G
MG
Brzozowski
A Marek
AM
Patkar
Shamkant A
SA
Vind
Jesper
J
Svendsen
Allan
A
Verger
Robert
R
D001203:Ascomycota / Q000201:enzymology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004735:Energy Transfer; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D005453:Fluorescence; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D008823:Micelles; D013379:Substrate Specificity
D005231:Fatty Acids, Unsaturated; D008823:Micelles; D008049:Lipase; C013641:parinaric acid