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

• Main Authors: Frank Peelman, Berlinda Vanloo, Jean-Luc Verschelde, Christine Labeur, Hans Caster, Josée Taveirne, Annick Verhee, Nicolas Duverger, Joël Vandekerckhove, Jan Tavernier, Maryvonne Rosseneu
• Format: Article
• Language: English
• Published: Elsevier 2001-04-01
• Series: Journal of Lipid Research
• Subjects: enzyme; lipase; structure; ionic interactions; phospholipid; lipoproteins
• Online Access: http://www.sciencedirect.com/science/article/pii/S0022227520303655

On the basis of structural homology calculations, we previously showed that lecithin:cholesterol acyltransferase (LCAT), like lipases, belongs to the α/β 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 proteins from different 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 reconstituted high density lipoprotein (rHDL) showed that mutations 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 α/β hydrolase fold and structural homology with pancreatic lipase observed 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 residues 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. Rosseneu. Effect of mutations of N- and C-terminal charged residues on the activity of LCAT. J. Lipid Res. 2001. 42: 471–479.