The structural and functional characterisation of 10 to 15 lipases. The aim of the project is to acquire so much new knowledge about a sufficient number of these enzymes that it will be possible to understand why they are lipases and how they function as such.
Research has been undertaken into the structure and catalytic mechanism of guinea pig pancreatic lipase (GPL) and mucor miehei lipase (MML). Results are as follows:
GPL has been purified to homogeneity;
partial sequence of GPL has been determined on a protein level;
GPL has been cloned;
the complete GPL complementary deoxyribonucleic acid (cDNA) sequence has been determined;
expression of recombinant GPL (rGPL) in Aspergillus has been achieved;
semilarge scale purification of rGPL has started;
preliminary crystallographic analysis of rGPL was started stereoselectivity of GLP and rGPL has been analyzed;
MML structure has been refined;
cocrystallization of MML with an inhibitor has been achieved;
data collection of MML inhibitor complex was finished;
model for interfacial activation has been produced;
stereoselectivity of MML has been analyzed.
The crystal structure of the mucor miehei inhibitor complex and the complete sequence of GPL have shown that the elucidation of the 3-dimensional structure around the active site (asparagine, histamine, serine) and around the lid is extremely important in understanding the mechanism of action of lipases.
The present study includes the determination of the 3-dimensional structure and catalytic mechanism of 2-3 triglyceride lipases of industrial relevance. The lipases studied are the fungal lipase from Mucor Miehei and the mammalian lipase from guinea pig pancreas.
In the case of the Mucor Miehei lipase, the 3-dimensional structure has previously been determined to a resolution of 2.0 angstroms. A more detailed understanding of the catalytic mechanism and conformation changes needed for enzyme action will be achieved by cocrystallization of the lipase with inhibitors and substrate analogues and subsequent determination of 3-dimensional structure of the lipase-inhibitor complex.
With respect to structural information, the guinea pig pancreatic triglyceride lipase is unknown in that neither the amino acid sequence nor the 3-dimensional structure of this enzyme is known. This lipase is interesting from a structure/function point of view, due to its ability, in contrast to other lipases, to hydrolyse phospholipids as well as triglyceride. As the enzyme has not previously been studied in detail, it will be necessary to isolate, clone and express a recombinant variant of guinea pig pancreatic lipase in order to obtain a sufficient amount of lipase for crystallization and 3-dimensional structure determination. The overall aim is to generate the 3-dimensional structure of guinea pig pancreatic lipase and to compare it with other mammalian lipases, eg the human pancreatic triglyceride lipases. In parallel with the structural studies, enzymatic studies on substrate specificity, interfacial kinetics and enzyme kinetics will be carried out with the overall aim of getting a detailed understand ng of the mechanism of hydrolysis.
Funding SchemeCSC - Cost-sharing contracts
YO1 5DD York