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Requirement of lid2 for interfacial activation of a family I.3 lipase with unique two lid structures
Author(s) -
Cheng Maria,
Angkawidjaja Clement,
Koga Yuichi,
Kanaya Shigenori
Publication year - 2012
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2012.08734.x
Subject(s) - lipase , triacetin , chemistry , substrate (aquarium) , enzyme , mutant , molecular dynamics , esterase , biophysics , biochemistry , stereochemistry , crystallography , biology , computational chemistry , ecology , gene
A family I.3 lipase from P seudomonas sp. MIS 38 ( PML ) is characterized by the presence of two lids (lid1 and lid2) that greatly change conformation upon substrate binding. While lid1 represents the commonly known lid in lipases, lid2 is unique to PML and other family I.3 lipases. To clarify the role of lid2 in PML , a lid2 deletion mutant (Δ L 2‐ PML ) was constructed by deleting residues 35–64 of PML . Δ L 2‐ PML requires calcium ions for both lipase and esterase activities as does PML , suggesting that it exhibits activity only when lid1 is fully open and anchored by the catalytically essential calcium ion, as does PML . However, when the enzymatic activity was determined using triacetin, the activity of PML exponentially increased as the substrate concentration reached and increased beyond the critical micellar concentration, while that of Δ L 2‐ PML did not. These results indicate that PML undergoes interfacial activation, while Δ L 2‐ PML does not. The activities of Δ L 2‐ PML for long‐chain triglycerides significantly decreased while its activity for fatty acid ethyl esters increased, compared with those of PML . Comparison of the tertiary models of Δ L 2‐ PML in a closed and open conformation, which are optimized by molecular dynamics simulation, with the crystal structures of PML suggests that the hydrophobic surface area provided by lid1 and lid2 in an open conformation is considerably decreased by the deletion of lid2. We propose that the hydrophobic surface area provided by these lids is necessary to hold the micellar substrates firmly to the active site and therefore lid2 is required for interfacial activation of PML . Database Triacylglycerol lipase ( EC 3.1.1.3 )