STRUCTURE OF BOVINE PANCREATIC CHOLESTEROL ESTERASE AT 1.6 ANGSTROM -NOVEL STRUCTURAL FEATURES INVOLVED IN LIPASE ACTIVATION

The structure of pancreatic cholesterol esterase, an enzyme that hydro lyzes a wide variety of dietary lipids, mediates the absorption of cho lesterol esters, and is dependent on bile salts for optimal activity, is determined to 1.6 Angstrom resolution. A full-length construct, mut ated to eliminate two N-linked glycosylation sites (N187Q/N361Q), was expressed in HEK 293 cells. Enzymatic activity assays show that the pu rified, recombinant, mutant enzyme has activity identical to that of t he native, glycosylated enzyme purified from bovine pancreas. The muta nt enzyme is monomeric and exhibits improved homogeneity which aided i n the growth of well-diffracting crystals. Crystals of the mutant enzy me grew in space group C2, with the following cell dimensions: a = 100 .42 Angstrom, b = 54.25 Angstrom, c = 106.34 Angstrom, and beta = 104. 12 degrees, with a monomer in the asymmetric unit. The high-resolution crystal structure of bovine pancreatic cholesterol esterase (R-cryst = 21.1%; R-free = 25.0% to 1.6 Angstrom resolution) shows an alpha-bet a hydrolase fold with an unusual active site environment around the ca talytic triad. The hydrophobic C terminus of the protein is lodged in the active site, diverting the oxyanion hole away from the productive binding site and the catalytic Ser194. The amphipathic, helical lid fo und in other triglyceride lipases is truncated in the structure of cho lesterol esterase and therefore is not a salient feature of activation of this lipase. These two structural features, along with the bile sa lt-dependent activity of the enzyme, implicate a new mode of lipase ac tivation.