Mk. Bhat et al., MODIFICATION OF THE HEADGROUP SELECTIVITY OF PORCINE PANCREATIC PHOSPHOLIPASE-A(2) BY PROTEIN ENGINEERING, Biochemistry, 32(45), 1993, pp. 12203-12208
On the basis of the three-dimensional structures of phospholipid and p
orcine pancreatic phospholipase A2(pla2), it was predicted that the re
moval of a negative charge in the hydrophilic region of the phospholip
id binding site would influence the head-group selectivity of porcine
pancreatic pla2. To test this prediction, glutamic acid 46 was changed
to leucine by site-directed mutagenesis. The E46L mutant, expressed i
n Escherichia coli, was purified and characterized. The mutation did n
ot affect the activity toward the mixed micellar substrate, but the ac
tivity of E46L toward DiC-12-P, which has two negative charges on the
head group, was three times higher than that of DiC-12-PC, which carri
es no net charge in the head group. The native pla2 was inhibited by t
he product(s) released from DiC-12-P but not the mutant enzyme. Kineti
c analysis revealed that the E46L mutant and the native pla2 had compa
rable affinities (K(m)) toward monomeric and micellar phospholipids of
zwitterionic type while the activity (k(cat)) of E46L, toward the sam
e substrates, was approximately 50% lower compared to that of native p
la2. When micellar DiC-12-P was used as a substrate, the K(m)app value
for E46L was four times lower and the k(cat)app/K(m)app was 5-fold hi
gher than those of native pla2. However, the kinetic parameters of mut
ant and native pla2s remained unchanged for monomeric HEPG, with one n
egative charge in the head group. Thus, we have modified the head-grou
p selectivity of porcine pancreatic pla2 by protein engineering.