AMINO-ACID RESIDUE-149 OF LECITHIN-CHOLESTEROL ACYLTRANSFERASE DETERMINES PHOSPHOLIPASE A(2) AND TRANSACYLASE FATTY ACYL SPECIFICITY

Human LCAT prefers phosphatidylcholine (PC) with sn-1-palmitoyl-2-oleo yl PC (POPC) as substrate for cholesteryl ester synthesis, whereas rat LCAT (which is 92% similar in amino acid sequence) prefers sn-1-palmi toyl-2-arachidonoyl PC (PAPC), Six recombinant human LCAT cDNA clones were constructed with unique clusters of rat sequence substitutions in the human background spanning the region encoding amino acids 121-296 , Media from transfected COS cells expressing each of the constructs w ere assayed for LCAT cholesterol esterification (CE) or phospholipase A(2) (PLA(2)) activity using substrate particles containing POPC or PA PC, The PAPC/POPC CE activity ratio of the cluster 1 construct (amino acids 149-158) was 1.3, resembling rat LCAT, whereas cluster 2-5 clone s produced CE activity ratios <0.3, unchanged from human LCAT, The clu ster 6 clone (Y292H/W294F) had an intermediate ratio (0.6), Similar re sults were observed for LCAT PLA(2) activity, In additional studies, p osition 149 of human LCAT was changed to the rat sequence (hE149A) and compared to a triple mutation containing the remainder of the cluster 1 changes (G151R/E154D/R158Q), CE and PLA(2) activity ratio for the h E149A construct was >1.7, similar to rat LCAT, whereas the triple muta tion construct retained a ratio similar to human LCAT (<0.6). Thus, a single amino acid substitution (E149A) was sufficient to alter the fat ty acyl specificity of human LCAT to that of rat LCAT, with an increas e in activity toward PAPC, This is the first example of a point mutati on in an enzyme with PLA(2) activity that results in an increase in ac tivity toward arachidonic acid.