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J. Biol. Chem., Vol. 264, Issue 25, 14723-14728, Sep, 1989
FS Bonelli and A Jonas
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.
Reaction of lecithin cholesterol acyltransferase with water-soluble substrates
Department of Biochemistry, College of Medicine, University of Illinois, Urbana 61801.
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