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PDBsum entry 1vap
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Lipid degradation
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PDB id
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1vap
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Structural aspects of interfacial adsorption. A crystallographic and site-Directed mutagenesis study of the phospholipase a2 from the venom of agkistrodon piscivorus piscivorus.
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Authors
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S.K.Han,
E.T.Yoon,
D.L.Scott,
P.B.Sigler,
W.Cho.
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Ref.
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J Biol Chem, 1997,
272,
3573-3582.
[DOI no: ]
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PubMed id
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Abstract
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Recent genetic and structural studies have shed considerable light on the
mechanism by which secretory phospholipases A2 interact with substrate
aggregates. Electrostatic forces play an essential role in optimizing
interfacial catalysis. Efficient and productive adsorption of the Class I bovine
pancreatic phospholipase A2 to anionic interfaces is dependent upon the presence
of two nonconserved lysine residues at sequence positions 56 and 116, implying
that critical components of the adsorption surface differ among enzyme species
(Dua, R., Wu, S.-K., and Cho, W. (1995) J. Biol. Chem. 270, 263-268). In an
effort to further characterize the protein residues involved in interfacial
catalysis, we have determined the high resolution (1.7 A) x-ray structure of the
Class II Asp-49 phospholipase A2 from the venom of Agkistrodon piscivorus
piscivorus. Correlation of the three-dimensional coordinates with kinetic data
derived from site-directed mutations near the amino terminus (E6R, K7E, K10E,
K11E, and K16E) and the active site (K54E and K69Y) defines much of the
interface topography. Lysine residues at sequence positions 7 and 10 mediate the
adsorption of A. p. piscivorus phospholipase A2 to anionic interfaces but play
little role in the enzyme's interaction with electrically neutral surfaces or in
substrate binding. Compared to the native enzyme, the mutant proteins K7E and
K10E demonstrate comparable (20-fold) decreases in affinity and catalysis on
polymerized mixed liposomes of
1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine and
1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol, while the double
mutant, K7E/K10E, shows a more dramatic 500-fold decrease in catalysis and
interfacial adsorption. The calculated contributions of Lys-7 and Lys-10 to the
free energy of binding of A. p. piscivorus phospholipase A2 to anionic liposomes
(-1.8 kcal/mol at 25 degrees C per lysine) are additive (i.e. -3.7 kcal/mol) and
together represent nearly half of the total binding energy. Although both lysine
side chains lie exposed at the edge of the proposed interfacial adsorption
surface, they are geographically remote from the corresponding 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. lysines) is species-specific. This
variability in the topography of the adsorption surface suggests a corresponding
flexibility in the orientation of the active enzyme at the substrate interface.
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Figure 2.
Fig. 2. A stereoview of the  -carbon
trace of the crystalline App-D49 indicating the positions of
mutated residues. The view of the enzyme shown here is similar
to that used in previous publications to illustrate the location
of a co-crystallized transition-state^ analog (9, 17, 28). The
active site lies at the base of^ the central cavity formed from
the amino-terminal helix, residues 19-23, portions of the
calcium-binding loop, and the side chain of Lys-69 and is
indicated by the side chain of His-48 (in black). The plane of
the putative interfacial adsorption surface lies perpendicular
to the hydrophobic channel and incorporates residues surrounding
the external opening of the channel. In the present study,
specific lysine residues (Lys-7, Lys-10, Lys-11, Lys-16, Lys-54,
and Lys-69) were changed into glutamates and tyrosine^ (Lys-69)
in an effort to characterize the structural determinants of
interfacial adsorption.
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Figure 5.
Fig. 5. The interaction of a transition-state analog
(L-1-O-octyl-2-heptylphosphonyl-sn-glycero-3-phosphoethanolamine)
with the^ active site of the Class I PLA[2] from the venom of N.
n. atra (A). Class II PLA[2]s, including App-D49, substitute a
lysine residue^ for the tyrosine at sequence position 69. The
K69Y mutant has essentially the same activity as the wild type
enzyme toward PC^ and PE substrates but shows a 3-fold drop in
activity toward PG substrate. One explanation for this finding
is that the  -ammonium
group of Lys-69 forms additional hydrogen bonds with
phospholipid^ head groups, especially with PG whose hydroxyl
groups can function as hydrogen bond acceptors (B). Such an
interaction would not be achievable by the phenolic oxygen of
Tyr-69 or with PC and^ PE as substrate.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(1997,
272,
3573-3582)
copyright 1997.
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