Interfacial catalysis: the mechanism of phospholipase A2

Abstract

A chemical description of the action of phospholipase A2 (PLA2) can now be inferred with confidence from three high-resolution x-ray crystal structures. The first is the structure of the PLA2 from the venom of the Chinese cobra (Naja naja atra) in a complex with a phosphonate transition-state analogue. This enzyme is typical of a large, well-studied homologous family of PLA2S. The second is a similar complex with the evolutionarily distant bee-venom PLA2. The third structure is the uninhibited PLA2 from Chinese cobra venom. Despite the different molecular architectures of the cobra and bee-venom PLA2s, the transition-state analogue interacts in a nearly identical way with the catalytic machinery of both enzymes. The disposition of the fatty-acid side chains suggests a common access route of the substrate from its position in the lipid aggregate to its productive interaction with the active site. Comparison of the cobra-venom complex with the uninhibited enzyme indicates that optimal binding and catalysis at the lipid-water interface is due to facilitated substrate diffusion from the interfacial binding surface to the catalytic site rather than an allosteric change in the enzyme's structure. However, a second bound calcium ion changes its position upon the binding of the transition-state analogue, suggesting a mechanism for augmenting the critical electrophile.

Fig. 1

(A) The transition-state analogue, L- -l-O-octyl-2-heptyl-phosphonyl-sn -glycero-3-phosphoethanolamine, is a short-chain member of a class of strong PLA₂ inhibitors studied by Gelb and co-workers (31, 32) in which a phosphonate replaces the sn-2 ester. This analogue was designed to emulate the transition state (B) formed in the hydrolysis of l,2-dioctanoyl-sn-3-phosphoethanolamine (C) and is therefore designated as diC₈(2Ph)PE. The conformation of diC₈(2Ph)PE as seen in the complex with the PLA₂ from N. n. atra venom was used to model the structure of l,2-dioctanoyl-sn-3-phosphoethanolamine and its transition state; the ester carbonyls are shown with correct bond distances and interbond angles but their positions do not necessarily represent the minimum energy conformations. (C, P, O, and N atoms are represented by black spheres, large open spheres, small open spheres, and striped spheres, respectively; the spheres with asterisks denote the positions of the oxyanions.)

Fig. 2

The structure of N. n. atra PLA₂; electron density map (2F_o–F_c) of the “attacking” water molecule from which His48 abstracts a proton (33). The enzyme was purified from crude venom (Miami Serpentarium) (32). Droplets (12 μl) containing 10 mg/ml protein, 10 mM CaCl₂, 0.1 M sodium cacodylate, 20 percent 1,4-butanediol, pH 6.8, were plated onto plastic cover slips and inverted over 1-ml reservoirs containing 0.1 M sodium cacodylate, 50 percent 1,4-butanediol, and 10 percent methyl alcohol. The crystals (0.4 mm by 0.2 mm by 0.2 mm) were of space group I4, a = b = 93.0 Å, and c = 22.2 Å, with one molecule in the asymmetric unit. Data to 1.5 Å resolution (R_sym = 0.05) were collected with two San Diego Multiwire System detectors; source radiation was graphite-monochromated CuK_α, emission from a Rigaku 300 x-ray generator. The structure was solved by molecular replacement with the program Merlot 1.5 (34) modified as described (35). The search molecule was the model of the same enzyme derived from the crystal structure of its complex with a phosphonate transition-state analogue (2). Refinement converged readily to an R factor of 0.143 for data in the resolution range 8.3 to 1.5 Å (F > 1.5 σ_F). On average, bond lengths, bond angle distances, and planarity deviated from ideal values by less than 0.016, 0.031, and 0.021 Å, respectively.

Fig. 3

Catalytic mechanism. Residues of the Class I/II superfamily are used here for familiarity. They are replaced by Asp67, His34, and Tyr87 in the bee-venom enzyme. Although Tyr52 and Tyr72 are absolutely conserved in the Class I/II homologous superfamily they are not shown in (B) and (C). (A) Catalytic attack on substrate bound in a productive mode. The positions of the protons are not known but assumed from other catalytic systems (36). (B) The tetrahedral intermediate as it collapses into products. (C) The products formed by “productive collapse.” Three water molecules move into the active site (as indicated by the arrows) to replace the products. One will engage the Nδ1 of His48, and the remaining two will coordinate the calcium ion (one equatorially and one axially).

Fig. 4

Interface between the transition-state analogue and the catalytic surface. (A) Naja naja atra–venom PLA₂; the catalytic surface in the absence of diC₈(2Ph)PE. Waters are labeled W, the primary calcium ion is labeled Ca, and the attacking nucleophile is cross-hatched. (B) Naja naja atra–venom PLA₂ (asymmetric unit “b”); P2 simulates the tetrahedral intermediate and P3 corresponds to the sn-3 phosphate. The interaction in asymmetric unit “a” is identical, as is the bee-venom interface where Asp49(35) contributes the carboxylate; residues 28(8), 30(10), and 32(12) of the calcium-binding loop contribute backbone carbonyls to the calcium ligation cage. Atoms are identified as in (A).

Fig. 5

Complexes of diC₈(2PH)PE with PLA₂s. (A and B) Naja naja atra–venom PLA₂. (A) Van der Waal’s surface showing the alkyl substituents of the phospholipid protruding from the surface (red) and surrounded in green by residues that form the “mouth” of the hydrophobic channel [Leu(Val)2, Phe5, Tyr6, Ile9, and Trp19] and by residues that have been shown to be [Tyr(Trp)3 and Trp19] or arc very likely to be [Lys6 and Arg31] part of the interfacial binding surface. The non-alkyl inhibitor elements are colored blue. (B) A skeletal representation of (A); the primary calcium ion is shown in yellow. (C and D) Bee-venom PLA₂. (C) Van der Waal’s surface color-coded as above except that the residues of the mouth of the channel and the interfacial binding surface (green) are surmised from their proximity to the alkyl substituents or the enzyme surface or both [Ile1, Tyr3, Cys9, His11, Thr56, Arg57, Leu59, Val83, Met86, Tyr87, and Ile91]. (D) A skeletal representation of (C); the primary calcium ion is shown in yellow.

Fig. 6

Schematic rendition of a productive interaction between a PLA₂ and aggregated substrate.