Bi. Lee et al., ROLES OF SURFACE HYDROPHOBIC RESIDUES IN THE INTERFACIAL CATALYSIS OFBOVINE PANCREATIC PHOSPHOLIPASE A(2), Biochemistry, 35(13), 1996, pp. 4231-4240
The interfacial binding is a unique and important step in the phosphol
ipase A(2) (PLA(2)) catalyzed hydrolysis of phospholipids which is dis
tinct from the binding of a substrate to the active site. To assess th
e roles of surface hydrophobic residues of PLA(2) in these processes,
we selectively mutated Leu-19 and Leu-20 of bovine pancreatic PLA(2) t
o charged (L19K and L20K), uncharged polar (L19S and L20S), and amphip
hilic (L19W and L20W) groups and measured their kinetic and binding pr
operties using various phospholipid aggregates, including micelles, mo
nolayers, and polymerized mixed liposomes. The mutations of Leu-19 and
Leu-20 did not significantly change either the tertiary structure or
the thermodynamic stability of bovine pancreatic PLA(2). Toward monome
ric 1,2-dihexanoyl-sn-glycero-3-phosphocholine, all Leu-20 mutants (L2
0S, L20W, and L20K) showed activities comparable to that of wild type
whereas the substitution of Leu-19 with less hydrophobic side chains (
L19S and L19K) reduced the activity to 70% and 50%. Toward zwitterioni
c 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC(8)PC) micelles, L20S
and L20K mutants showed only 30% and 351e of the wild-type activity,
respectively, whereas L20W was about twice as active as wild type. Als
o, L19S and L19K showed 75% and 15% of the wild-type activity. respect
ively. Toward anionic Triton X-100/sodium deoxycholate/diC(8)PC (4:2:1
) mixed micelles, L20W and L20K were 2.6 times and twice more active t
han wild type. To determine the sn-2 acyl group selectivity of wild ty
pe and mutants, polymerized mixed liposomes were used which contained
ipoyloxy)-dodecanoyl]-sn-glycero-3-phosphoglycerol and 1 mol % of eith
er dodecanoyl]-2-hexanoyl-sn-glycero-3-phosphocholine or ecanoyl]-2-do
decanoyl-sn-glycero-3-phosphocholine. These measurements showed that L
eu-19 was involved in the substrate binding and the sn-2 acyl group se
lectivity of bovine pancreatic PLA(2) and that Leu-20 made a direct co
ntact with the surface of phospholipid aggregates. The binding affinit
ies of mutants to micelles, polymerized liposomes, and monolayers were
well consistent with their kinetic behaviors, supporting the notion t
hat the altered activities of Leu-19 mutants and Leu-20 mutants were d
ue to the change in their substrate binding and interfacial binding, r
espectively. Finally, the L20W mutant represents the first example of
protein engineering of PLA(2) which results in a significant increase
in interfacial binding to densely packed neutral monolayers and bilaye
rs.