Phospholipase A2 (group IB)

 

PLA2 catalyses the hydroylsis of the 2-acyl ester of 1,2-diacylphosphatides. Two forms of PLA2 are known: the small secretory PLA2s which are amphipathic molecules usually found associated with lipid membranes, and the larger cytosolic forms. This annotation is for the small secretory form. Secretory PLA2s form part of the neurotoxic component of many snake and bee venoms due to its ability to block acetylcholine release.

Secretory PLA2s have been divided into 3 main groups: I, II, and III. Class I (present in some snake venoms and in mammalian exocrine pancrease) and II (present in some snake venoms and also broadly distributed among a variety of mammalian cell types) are closely related to each other. The class III enzymes (including venom enzymes from the honeybee and the Gila monster) appear to form a separate divergent group though their active site and mechanism is similar. The key catalytic histidine and aspartate are in very similar orientations however the residues making up the rest of the hydrogen bond network are not conserved spatially.

 

Reference Protein and Structure

Sequence
P00592 UniProt (3.1.1.4) IPR001211 (Sequence Homologues) (PDB Homologues)
Biological species
Sus scrofa (pig) Uniprot
PDB
1l8s - CARBOXYLIC ESTER HYDROLASE COMPLEX (DIMERIC PLA2 + LPC-ether + ACETATE + PHOSPHATE IONS) (1.55 Å) PDBe PDBsum 1l8s
Catalytic CATH Domains
1.20.90.10 CATHdb (see all for 1l8s)
Cofactors
Calcium(2+) (1) Metal MACiE

Enzyme Reaction (EC:3.1.1.4)

1,2-diacyl-sn-glycero-3-phosphocholine
CHEBI:57643ChEBI
+
water
CHEBI:15377ChEBI
→
1-O-acyl-sn-glycero-3-phosphocholine
CHEBI:58168ChEBI
+
fatty acid anion
CHEBI:28868ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Lecithinase A, Phosphatidase, Phosphatidolipase, Phospholipase A, Phospholipase A2,

Enzyme Mechanism

Introduction

A standard numbering system exists for class I and class II enzymes; in this system the catalytic residues are Gly 30, His 48, and Asp 99.

The reaction involves attack by a water molecule on the ester carbonyl to give a tetrahedral intermediate which then collapses with loss of the alcoholate leaving group. A Ca(II) ion and the backbone NH of Gly 30 form an oxyanion hole to stabilise negative charge on the tetrahedral intermediate. There has been some debate however on exactly how the nucleophilic water molecule is activated, see references PMID:11749391 or PMID:12501175 for a discussion. In one proposed mechanism, His 47 deprotonates the attacking water molecule and later protonates the departing alcoholate leaving group. Asp 99 functions to modify the pKa of His 48 as in the serine proteases. A second proposed mechanism involves two water molecules at the active site. One of these (w6) deprotonates the other (w5) and is itself deprotonated by His 48, while w5 attacks the ester carbonyl. The departing alcoholate leaving group is protonated by w6 which is itself reprotonated by His 48. In this second proposed mechanism, which has been used for this annotation, the Ca2+ ion coordinates w5 and lowers its pKa as well as stabilising the tetrahedral intermediate.

The existence of a second Ca(II) ion in stabilising the tetrahedral intermediate has also been proposed, see references PMID:8203286 and PMID:1201175. This is suggested to interact with the amide oxygen of the peptide bond between residue 29 and Gly 30, hyperpolarising this peptide bond and so increasing oxyanion stabilisation by the NH group of Gly 30. However some sPLA2 enzymes do not seem to contain this second calcium ion or even a potential coordination site for it.

Catalytic Residues Roles

UniProt PDB* (1l8s)
Gly52 (main-N) Gly30B (main-N) Backbone amide forms part of the oxyanion hole that stabilises the tetrahedral intermediate resulting from nucleophilic attack by water on the substrate carbonyl. hydrogen bond donor, electrostatic stabiliser
Tyr95, Tyr74 Tyr73B, Tyr52B Help stabilise and activate the Asp99 residue. hydrogen bond donor, electrostatic stabiliser
Asp121 Asp99B Asp forms a hydrogen bond with the Ne2 of the catalytic His and neutralises the positive charge of this His created by the ester bond cleavage during catalysis. hydrogen bond acceptor, electrostatic stabiliser
His70 His48B The catalytic His is hydrogen bonded to an active site water via the His N-delta-1 atom. His abstracts a proton from this water molecule, activating it towards nucleophilic attack of the carbonyl carbon of the substrate. The histidine is also hydrogen bonded to the catalytic Asp, forming the catalytic dyad. His 64 interacts electrostatically with the Ca(II) cofactor for stabilisation. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
Asp71, Gly52 (main-C), Tyr50 (main-C), Gly54 (main-C) Asp49B, Gly30B (main-C), Tyr28B (main-C), Gly32B (main-C) Forms part of the Ca(II) binding site. metal ligand
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic addition, overall reactant used, intermediate formation, unimolecular elimination by the conjugate base, overall product formed, native state of enzyme regenerated, intermediate collapse, intermediate terminated

References

  1. Scott DL et al. (1994), Adv Protein Chem, 45, 53-88. Structure and Catalytic Mechanism of Secretory Phospholipases A2. DOI:10.1016/s0065-3233(08)60638-5. PMID:8154374.
  2. Madsen JJ et al. (2011), J Phys Chem B, 115, 6853-6861. Secretory Phospholipase A2Activity toward Diverse Substrates. DOI:10.1021/jp112137b. PMID:21561115.
  3. Leopoldini M et al. (2010), J Phys Chem B, 114, 11584-11593. Favored Reaction Mechanism of Calcium-Dependent Phospholipase A2. Insights from Density Functional Exploration. DOI:10.1021/jp1003819. PMID:20718411.
  4. Linderoth L et al. (2008), Biophys J, 94, 14-26. Molecular Basis of Phospholipase A2 Activity toward Phospholipids with sn-1 Substitutions. DOI:10.1529/biophysj.107.110106. PMID:17827229.
  5. Sekar K et al. (2006), Acta Crystallogr D Biol Crystallogr, 62, 717-724. Suggestive evidence for the involvement of the second calcium and surface loop in interfacial binding: monoclinic and trigonal crystal structures of a quadruple mutant of phospholipase A2. DOI:10.1107/s0907444906014855. PMID:16790927.
  6. Poi MJ et al. (2003), J Mol Biol, 329, 997-1009. A Low-barrier Hydrogen Bond Between Histidine of Secreted Phospholipase A2 and a Transition State Analog Inhibitor. DOI:10.1016/s0022-2836(03)00512-6.
  7. Pan YH et al. (2002), Biochemistry, 41, 14790-14800. Crystal Structure of Phospholipase A2Complex with the Hydrolysis Products of Platelet Activating Factor:  Equilibrium Binding of Fatty Acid and Lysophospholipid-Ether at the Active Site May Be Mutually Exclusive†,‡. DOI:10.1021/bi026922r. PMID:12475227.
  8. Edwards SH et al. (2002), Biochemistry, 41, 15468-15476. The Crystal Structure of the H48Q Active Site Mutant of Human Group IIA Secreted Phospholipase A2at 1.5 Å Resolution Provides an Insight into the Catalytic Mechanism†,‡. DOI:10.1021/bi020485z. PMID:12501175.
  9. Berg OG et al. (2001), Chem Rev, 101, 2613-2654. Interfacial Enzymology:  The Secreted Phospholipase A2-Paradigm. DOI:10.1021/cr990139w. PMID:11749391.
  10. Yuan C et al. (1999), Biochim Biophys Acta Mol Cell Biol Lipids, 1441, 215-222. Pancreatic phospholipase A2: new views on old issues. DOI:10.1016/s1388-1981(99)00156-0.
  11. Segelke BW et al. (1998), J Mol Biol, 279, 223-232. Structures of two novel crystal forms of Naja naja naja phospholipase A2 lacking Ca2+ reveal trimeric packing. DOI:10.1006/jmbi.1998.1759. PMID:9636712.
  12. Zhao H et al. (1998), Toxicon, 36, 875-886. Structure of a snake venom phospholipase A2 modified by p-bromo-phenacyl-bromide. DOI:10.1016/s0041-0101(97)00169-4. PMID:9663694.
  13. Sekar K et al. (1997), Biochemistry, 36, 3104-3114. Phospholipase A2Engineering. Structural and Functional Roles of the Highly Conserved Active Site Residue Aspartate-99†. DOI:10.1021/bi961576x. PMID:9115986.
  14. Sekar K et al. (1997), Biochemistry, 36, 14186-14191. Crystal Structure of the Complex of Bovine Pancreatic Phospholipase A2with the Inhibitor 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol†,‡. DOI:10.1021/bi971370b. PMID:9369492.
  15. Cha SS et al. (1996), J Med Chem, 39, 3878-3881. High-Resolution X-ray Crystallography Reveals Precise Binding Interactions between Human Nonpancreatic Secreted Phospholipase A2and a Highly Potent Inhibitor (FPL67047XX). DOI:10.1021/jm960502g. PMID:8831753.
  16. Scott DL et al. (1994), Adv Inorg Biochem, 10, 139-155. The structural and functional roles of calcium ion in secretory phospholipases A2. PMID:8203286.
  17. Scott DL et al. (1990), Science, 250, 1563-1566. Crystal structure of bee-venom phospholipase A2 in a complex with a transition-state analogue. DOI:10.1126/science.2274788. PMID:2274788.
  18. Dijkstra BW et al. (1983), J Mol Biol, 168, 163-179. Structure of porcine pancreatic phospholipase A2 at 2.6 A resolution and comparison with bovine phospholipase A2. PMID:6876174.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, James W. Murray, Atlanta Cook, Craig Porter