Z. Shen et al., EFFECTS OF SPECIFIC FATTY-ACID ACYLATION OF PHOSPHOLIPASE A(2) ON ITSINTERFACIAL BINDING AND CATALYSIS, Biochemistry, 33(38), 1994, pp. 11598-11607
Monomeric phospholipase A(2) (PLA(2)) from the venom of Agkistrodon pi
scivorus piscivorus (App-D49) was treated with 3-acyloxy-4-nitrobenzoi
c acids to acylate the epsilon-amino groups of two lysines (Lys-7 and
Lys-10) in the amino terminal region. Resulting 7,10-diacylated-App-D4
9s, with acyl groups ranging from lauroyl to palmitoyl, spontaneously
aggregated in solution. By contrast, 7,10-dioctanoyl-App-D49 existed a
s a monomer under the same condition. Kinetic and interfacial binding
properties of diacylated enzymes indicated that they catalyzed the hyd
rolysis at the interface as a monomer. When compared to nonacylated Ap
p-D49, diacylated enzymes showed slightly increased activity or decrea
sed activity toward monodispersed 1,2-dibutyryl-sn-glycero-3-phosphoch
olin Triton X-100/1,2-dilauroyl-sn-glycero-3-phosphocholine mixed mice
lles, and small unilamellar vesicles (SUV) of 1-palmitoyl-2-oleoyl-sn-
glycero-3-phosphochorine (POPC). Toward densely-packed liquid-crystall
ine phospholipid bilayers, such as large unilamellar vesicles (LUV) of
POPC, however, diacylated enzymes exhibited a large increase in activ
ity, which reached up to 250-fold for 7,10-dilauroyl-App-D49 ((k(cat)/
K-m)(app) = (1.0 +/- 0.02) x 10(6) M(-1) s(-1)). Measurements of the p
enetration of individual diacylated enzymes into 2-oleoyl-3-palmitoyl-
sn-glycero-1-phosphochorine (i.e., D-POPC) monolayers indicated that t
he acyl groups enhanced the interfacial binding of protein by interact
ing with hydrocarbon moieties of phospholipids and that these hydropho
bic interactions remained effective even when the phospholipid packing
density was high. Furthermore, fluorometric measurements of the bindi
ng of diacylated enzymes to polymerized vesicles of (lipoyloxy)dodecan
oyl]-sn-glycero-3-phosphocholine showed that the hydrophobic interacti
ons increased the enzymatic activity toward LUV by accelerating the mi
gration of enzyme molecules to vesicle surfaces. The analysis of the k
inetic course of POPC LUV hydrolysis showed that diacylated enzymes as
a catalyst were superior to nonacylated App-D49 in that they were not
only more catalytically efficient but also able to catalyze more turn
overs without being trapped in product-containing vesicles. In summary
, the acylation of App-D49 by 3-acyloxy-4-nitrobenzoic acids provides
a simple and convenient way of converting the enzyme into a highly act
ive form toward densely-packed liquid-crystalline phospholipid bilayer
s, which might have potential industrial and biomedical applications.