The Enzymatic Activity of Lipases Correlates with Polarity-Induced Conformational Changes: A Trp-Induced Quenching Fluorescence Study

Abstract

Triacylglycerol hydrolases (EC 3.1.1.3) are thought to become activated when they encounter the water-lipid interface causing a "lid" region to move and expose the catalytic site. Here, we tested this idea by looking for lid movements in Thermomyces lanuginosus lipase (TL lipase), and in variants with a mutated lid region of esterase (Esterase) and esterase/lipase (Hybrid) character. To measure lid movements, we employed the tryptophan-induced quenching (TrIQ) fluorescence method to measure how effectively a Trp residue on the lid of these mutants (at position 87 or 89) could quench a fluorescent probe (bimane) placed at nearby site 255 on the protein. To test if lid movement is induced when the enzyme detects a lower-polarity environment (such as at the water-lipid interface), we performed these studies in solvents with different dielectric constants (ε). The results show that lid movement is highly dependent on the particular lid residue composition and solvent polarity. The data suggest that in aqueous solution (ε = 80), the Esterase lid is in an "open" conformation, whereas for the TL lipase and Hybrid, the lid remains "closed". At lower solvent polarities (ε < 46), the lid region for all of the mutants is more "open". Interestingly, these behaviors mirror the structural changes thought to take place upon activation of the enzyme at the water-lipid interface. Together, these results support the idea that lipases are more active in low-polarity solvents because the lid adopts an "open" conformation and indicate that relatively small conformational changes in the lid region play a key role in the activation mechanism of these enzymes.

Identification of a pre-active conformation of a pentameric channel receptor

“…Bimane-133 shows a marked blue shift (15 nm) and a fluorescence two times higher than the denatured protein at pH 7.3 possibly indicating the probe when reacted to position 133 is located in a confined/hydrophobic environment (Kosower et al, 1982; Skjold-Jørgensen et al, 2015) (Figure 3A and Figure 3—figure supplement 2). To generate quenching pairs, we introduced tryptophan residues on the adjacent subunit β-sandwich (Figure 1A).…”

The Lid Domain in Lipases: Structural and Functional Determinant of Enzymatic Properties

“…They also suggested the key role that the lid plays in determining the polarity-dependent activation of lipases using a combination of methods measuring enzymatic activity, detecting structural changes using the tryptophan-induced quenching method, and calculating the lid opening energies using an MD simulations, and suggested that mutagenesis of the lid can lower the energy barrier associated with lid opening (Skjold-Jorgensen et al, 2016). Tryptophan-induced quenching fluorescence method has been applied to successfully measure the lid movements in T. lanuginosus lipase and its variants in solvents with different dielectric constants (Skjold-Jorgensen et al, 2015). The results indicated that lid movement is highly dependent on the particular lid residue composition as well as solvent polarity.…”

Understanding the activation mechanism of Thermomyces lanuginosus lipase using rational design and tryptophan‐induced fluorescence quenching

“…Further studies have used similar protein engineering strategies in endothelial lipase (EL) , human pancreatic lipase (HPL) , C. rugusa lipase (CRL) , Rhizopus chinensis (RCL) lipase , Aspergillus niger lipase (ANL) . Recently, Skjold‐ Jørgensen et al altered the activation mechanism in Thermomyces lanugiunosus lipase (TLL) by rational design of the lid‐region . All these studies on structurally diverse lipases, suggest that the lid‐region plays a key role in hydrolytic activity, interfacial activation, and substrate specificity.…”