STRUCTURAL ASPECTS OF INTERFACIAL ADSORPTION - A CRYSTALLOGRAPHIC ANDSITE-DIRECTED MUTAGENESIS STUDY OF THE PHOSPHOLIPASE A(2) FROM THE VENOM OF AGKISTRODON PISCIVORUS PISCIVORUS
Sk. Han et al., STRUCTURAL ASPECTS OF INTERFACIAL ADSORPTION - A CRYSTALLOGRAPHIC ANDSITE-DIRECTED MUTAGENESIS STUDY OF THE PHOSPHOLIPASE A(2) FROM THE VENOM OF AGKISTRODON PISCIVORUS PISCIVORUS, The Journal of biological chemistry, 272(6), 1997, pp. 3573-3582
Recent genetic and structural studies have shed considerable light on
the mechanism by which secretory phospholipases A(2) interact with sub
strate aggregates. Electrostatic forces play an essential role in opti
mizing interfacial catalysis. Efficient and productive adsorption of t
he Class I bovine pancreatic phospholipase A(2) to anionic interfaces
is dependent upon the presence of two nonconserved lysine residues at
sequence positions 56 and 116, implying that critical components of th
e adsorption surface differ among enzyme species (Dua, R. Wu, S.-K., a
nd Cho, W. (1995) J. Biol. Chem. 270, 263-268). In an effort to furthe
r characterize the protein residues involved in interfacial catalysis,
we have determined the high resolution (1.7 Angstrom) x-ray structure
of the Class II Asp-49 phospholipase A(2) from the venom of Agkistrod
on piscivorus piscivorus, Correlation of the three-dimensional coordin
ates with kinetic data derived from site-directed mutations near the a
mino terminus (E6R, K7E, K1OE, K11E, and K16E) and the active site (K5
4E and K69Y) defines much of the interface topography, Lysine residues
at sequence positions 7 and 10 mediate the adsorption of A. p. pisciv
orus phospholipase A(2) to anionic interfaces but play Little role in
the enzyme's interaction with electrically neutral surfaces or in subs
trate binding. Compared to the native enzyme, the mutant proteins K7E
and K1OE demonstrate comparable (20-fold) decreases in affinity and ca
talysis on polymerized mixed liposomes of l-2-(1-pyrenedecanoyl)-sn-gl
ycero-3-phosphocholine and ipoyloxy)dodecanoyl]-sn-glycero-3-phosphogl
ycerol, while the double mutant, K7E/K1OE, shows a more dramatic 500-f
old decrease in catalysis and interfacial adsorption, The calculated c
ontributions of Lys-7 and Lys-10 to the free energy of binding of A. p
. piscivorus phospholipase A(2) to anionic liposomes (-1.8 kcal/mol at
25 degrees C per lysine) are additive (i.e. -3.7 kcal/mol) and togeth
er represent nearly half of the total binding energy, Although both ly
sine side chains lie exposed at the edge of the proposed interfacial a
dsorption surface, they are geographically remote from the correspendi
ng interfacial determinants for the bovine enzyme. Our results confirm
that interfacial adsorption is largely driven by electrostatic forces
and demonstrate that the arrangement of the critical charges (e.g. ly
sines) is species-specific. This variability in the topography of the
adsorption surface suggests a corresponding flexibility in the orienta
tion of the active enzyme at the substrate interface.