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 acyltrans- ferase (LCAT), like lipases, belongs to the a / b hydrolase fold family. As there is higher sequence conservation in the N-terminal region of LCAT, we investigated the contribu- tion 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 proteins from dif- ferent species, were mutated in the N-terminal (residues 1- 210) and C-terminal (residues 211-416) regions of LCAT. Measurements of LCAT-specific activity on a monomeric substrate, on low density lipoprotein (LDL), and on recon- stituted high density lipoprotein (rHDL) showed that muta- tions of N-terminal conserved basic residues affect LCAT activity more than those in the C-terminal region. This agrees with the highest conservation of the a / b hydrolase fold and structural homology with pancreatic lipase ob- served for the N-terminal region, and with the location of most of the natural mutants reported for human LCAT. The structural homology between LCAT and pancreatic lipase further suggests that residues R80, R147, and D145 of LCAT 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 resi- dues R80, D145, and R147 of LCAT are accompanied by a substantial decrease or loss of activity, suggesting that salt bridges between these residues might contribute to the structural stability of the enzyme. — Peelman, F., B. Vanloo, J-L. Verschelde, C. Labeur, H. Caster, J. Taveirne, A. Verhee, N. Duverger, J. Vandekerckhove, J. Tavernier, and M. Rosse- neu. Effect of mutations of N- and C-terminal charged resi- dues on the activity of LCAT. J. Lipid Res. 2001. 42: 471- 479.