Reaction of lecithin cholesterol acyltransferase with water-soluble substrates.

To separate the interfacial and catalytic reactions of lecithin cholesterol acyltransferase (LCAT), we carried out the first investigation of its reaction with water-soluble substrates. We used a continuous spectrophotometric assay for the hydrolysis of p-nitrophenyl esters of fatty acids to determine the chain length specificity of the enzyme and its modulation by anions and apolipoproteins in solution. By chemical modification of amino acid residues, we demonstrated that the active site serine and histidine residues participate in both the esterase and acyltransferase reactions but that cysteine residues are not involved in the esterase reaction. The kinetics of the LCAT reaction were measured for p-nitrophenyl esters of fatty acids having up to six (C-6) carbons in length. With increasing acyl chain lengths the optimal reaction rates occurred for the C-5 ester and Km and Vmax values decreased progressively, while the specificity constant, kcat/Km, increased. The same series of substrates and longer chain esters, up to C-16, were also reacted with LCAT in the presence of Triton X-100 in order to determine the general trends for the reaction rates as a function of chain length. The observed trends for the reaction rates and kinetic constants were attributed to an increasing binding affinity for the longer acyl chains in a large hydrophobic cavity, with a concomitant restriction in the motions of the substrates and a decreased probability for the correct positioning of the ester bond for hydrolysis, resulting in a decreased substrate turnover. Since the kinetics of the interfacial reactions of LCAT are very sensitive to the presence of anions and apolipoproteins, in particular apoA-I, we investigated the effects of these modulators on the reactions of LCAT in solution. Unlike the interfacial reactions, the hydrolysis of the p-nitrophenyl esters was not affected by 0.1 M concentrations of anions nor by water-soluble apolipoproteins (apoA-I, apoA-II, and apoCs). Thus the regulation of the activity of LCAT is mediated largely by the interfaces on which it acts.