Effect of mutations of N- and C-terminal charged residues on the activity of LCAT

On the basis of structural homology calculations, we previously showed that lecithin:cholesterol acyltransferase (LCAT), like lipases, belongs to the alpha/beta hydrolase fold family. As there is higher sequence conservation in the N-terminal region of LCAT, we investigated the contribution of the N - and C-terminal conserved basic residues to the catalytic activity of this enzyme. Most basic, and some acidic residues, conserved among LCAT protein s from different species, were mutated in the N-terminal (residues 1-210) a nd C-terminal (residues 211-416) regions of LCAT Measurements of LCAT-speci fic activity on a monomeric substrate, on low density lipoprotein (LDL), an d on reconstituted high density lipoprotein (rHDL) showed that mutations of N-terminal conserved basic residues affect LCAT activity more than those i n the C-terminal region. This agrees with the highest conservation of the a lpha/beta hydrolase fold and structural homology with pancreatic lipase obs erved for the N-terminal region, and with the location of most of the natur al mutants reported for human LCAT. The structural homology between LCAT an d pancreatic lipase further suggests that residues R80, R147, and D145 of L CAT might correspond to residues R37, K107, and D105 of pancreatic lipase, which form the salt bridges D105-K107 and D105-R37. Natural and engineered mutations at residues R80, D145, and R147 of LCAT are accompanied by a subs tantial decrease or loss of activity, suggesting that salt bridges between these residues might contribute to the structural stability of the enzyme.