PDBsum entry 1vap

Lipid degradation

PDB id: 1vap

Contents

Protein chains

123 a.a. *

Waters ×139

* Residue conservation analysis

PDB id:

1vap

Name:

Lipid degradation

Title:

The monomeric asp49 secretory phospholipase a2 from the venom of agkistridon piscivorus piscivorus

Structure:

Phospholipase a2. Chain: a, b. Other_details: secretory phospholipase a2

Source:

Agkistrodon piscivorus piscivorus. Eastern cottonmouth. Organism_taxid: 8716. Strain: piscivorus

Resolution:

1.60Å R-factor: 0.197

Authors:

D.L.Scott

Key ref:

S.K.Han et al. (1997). Structural aspects of interfacial adsorption. A crystallographic and site-directed mutagenesis study of the phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. J Biol Chem, 272, 3573-3582. PubMed id: 9013608 DOI: 10.1074/jbc.272.26.16152

Date:

15-Nov-96 Release date: 07-Jul-97

Protein chains

P51972  (PA2B1_AGKPI) -  Basic phospholipase A2 APP-D49 from Agkistrodon piscivorus piscivorus

Seq: 123 a.a.

Struc: 123 a.a.

Enzyme reactions

• Enzyme class: E.C.3.1.1.4 - phospholipase A2.
• Reaction: a 1,2-diacyl-sn-glycero-3-phosphocholine + H2O = a 1-acyl-sn-glycero-3- phosphocholine + a fatty acid + H⁺
• Cofactor: Ca(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.272.26.16152

J Biol Chem 272:3573-3582 (1997)

PubMed id: 9013608

Structural aspects of interfacial adsorption. A crystallographic and site-directed mutagenesis study of the phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus.

S.K.Han, E.T.Yoon, D.L.Scott, P.B.Sigler, W.Cho.

ABSTRACT

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.

Selected figure(s)

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.

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.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (1997, 272, 3573-3582) copyright 1997.

Figures were selected by an automated process.