|M.Sc Student||Berkovitch Yulia|
|Subject||Structural Elements in Lipase T6 that Affect Hydrolysis of|
Triglycerides at the sn-2 Position
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Yuval Shoham|
The aim of this research was to identify structural components of lipase T-6 (E.C. 22.214.171.124, triacylglycerol hydrolases) that determine regioselectivity towards its natural substrates. Most lipases show regioselectivity towards the primary position of triacylglycerols (sn-1 and/or sn-3 position), while few others exhibit random activity. Lipases with specific regioselectivity towards the sn-2 position of triglycerides have not been found yet in nature, and the structural elements that determine the regioselectivity are not well understood. To gain insight into the hydrolysis profile towards defined position of the triglyceride in lipase from the thermophilic bacterium Geobacillus stearothermophilus T-6, two 3D model structures were constructed. The first model represents the open conformation of the enzyme with a triglyceride-mimic ligand bound to the active site. The second model is in close conformation, without a substrate, where a mobile helix (lid) blocks the catalytic cleft to the solvent. Based on these models, several point mutations were performed. The total and positional hydrolysis activities of the wild type lipase and the various mutants were determined using pH-stat and TLC. Mutations at the active groove hydrophobic residues, that are distant from the catalytic site, did not change the positional preference. However, some of them cause a significant increase in the total lipolytic activity of the enzyme. Mutations at residues T61 and Y73, which form hydrogen bonds with the un-scissiled substrate chains, caused a significant decrease in total lipolytic activity, and had only small effect on the regioselectivity. Both residues bind the sn-2 chain of the triglyceride and apparently determine the distance among the ester carbonyls of the three chains, thus dictating the position of the substrate at the active site. Breaking even one of these two hydrogen bonds results in a decrease of regioselectivity toward the sn-1/3 positions and a decrease of the lipolytic activity. The best results were obtained with the mutant H156E, which yields 2-fold hydrolysis at the sn-2 position as compared to the wild type enzyme. Histidine 156 is located close to the catalytic residues in lipase T6, but is not involved in the catalytic mechanism.