Structural Basis for Low Catalytic Activity in Lys49 Phospholipases A2A Hypothesis:  The Crystal Structure of Piratoxin II Complexed to Fatty Acid^,

Abstract

Asp49 plays a fundamental role in supporting catalysis by phospholipases A2 by coordinating the calcium ion which aids in the stabilization of the tetrahedral intermediate. In several myotoxins from the venoms of Viperidae snakes, this aspartic acid is substituted by lysine. The loss of calcium binding capacity by these mutants has become regarded as the standard explanation for their greatly reduced or nonexistent phospholipolytic activity. Here we describe the crystal structure of one such Lys49 PLA2, piratoxin-II, in which a fatty acid molecule is observed within the substrate channel. This suggests that such toxins may be active enzymes in which catalysis is interrupted at the stage of substrate release. Comparison of the present structure with other PLA2s, both active and inactive, identifies Lys122 as one of the likely causes of the increased affinity for fatty acid in Lys49 enzymes. Its interaction with the mainchain carbonyl of Cys29 is expected to lead to hyperpolarization of the peptide bond between residues 29 and 30 leading to an increased affinity for the fatty acid headgroup. This strongly bound fatty acid may serve as an anchor to secure the toxin within the membrane thus facilitating its pathological effects.

The Crystal Structure of a Novel, Inactive, Lysine 49 PLA2 from Agkistrodon acutus Venom

“…For the enzymatic activity of Asp 49 PLA2s, Scott et al (41) proposed that the backbone amide N-H of residue Gly 30 plays a role in stabilizing the oxyanion of the substrate tetrahedral intermediate (41). On the other hand, for the enzymatic activity of Lys 49 PLA2s, Lee et al (9) demonstrated that the hyperpolarization of the peptide bond between residues 29 and 30, induced by the hydrogen interaction between the main-chain carbonyl of Cys 29 and Lys 122 via a water molecule, may increase the affinity for the enzyme to the reaction product (fatty acid). In acutohaemolysin, the poor density appearance and high average temperature factor of peptide segment Val 31 -Gly-Gly 33 indicate relatively high dynamics.…”

“…Furthermore, the crystal structures of two Lys 49 PLA2s complexed with their substrates reveal that these proteins have the ability to bind substrates to their active sites (8,42). It has been proposed that Lys 49 PLA2s possess interrupted catalytic activity, because the residue Lys 122 hyperpolarizes the peptide bond between residues Cys 29 and Gly 30 , resulting in failure to release the reaction product (9).…”

A Molecular Mechanism for Lys49-Phospholipase A2 Activity Based on Ligand-induced Conformational Change

“…"SequenceSpace" analysis (42) and x-ray diffraction studies (32)(33)(34)(43)(44)(45)(46) have both clearly shown that, with the exception of Asp 49 itself, the Lys 49 -PLA 2 s conserve all the important residues of the catalytic machinery, as well the nucleophilic water molecule and a hydrogen-bonding network that involves Tyr 52 and Tyr 73 and the N terminus. In addition, crystallographic structures in complex with naturally bound fatty acid molecules (interpreted as the product of catalysis) have been described (46,47). Based on these results, Lee and co-workers (47) have suggested that the apparent low level or lack of catalytic activity for Lys 49 -PLA 2 observed in vitro could be the result of the failure of product release from the active site after one cycle of catalysis, leading to enzyme inhibition.…”

Crystallization and Preliminary X-Ray Crystallographic Studies of a Myotoxic Lys49-phospholipase A2 from Bothrops jararacussu Venom Complexed with α-Tocopherol Inhibitor

“…This corresponds to a Matthews coefficient (Navaza AC 1994) [32] of approximately 2.1 Å 3 /Da for both native and complexed crystals, within the expected range for typical protein crystals (assuming a value of 0.74 cm 3 /g for the protein partial specific volume). The crystal structures were determined by molecular replacement techniques implemented in the program AMoRe (Jones CMMMD 1990) [33] using the coordinates of a monomer of native PrTX-II (Lee B 2001) [15]. An electron density fortocopherol was observed in the hydrophobic channel for both monomers of BthTX-I-T complex (Fig.…”