Phospholipase catalysis

Roberts et al.40 used bolaform phosphatidylcholine as a probe of water soluble phospholipase catalysis. These bolaphiles (Figure 14) contain two phosphatidylcholines, as the ionic head groups permitting the evaluation of the proposal that two phosphatidylcholines are required for phospholipase activity. Phospholipase activity was measured using micelles formed from these bolaphiles and phosphatidylcholine containing amphiphiles. Increased membrane stability of these bolaform... 

Okada S, Jelinek R, Charych D (1999) Induced color change of conjugated polymeric vesicles by interfacial catalysis of phospholipase A(2). Angew Chem Int Ed 38 655-659... 

MICELLAR SUBSTRATES. Phospholipids in micelles are frequently found to be more active substrates in lipolysis than those phospholipids residing in a lipid bilayer". Dennis first described the use of Triton X-100 to manipulate the amount of phospholipid per unit surface area of a micelle in a systematic analysis of the interfacial interactions of lipases with lipid micelles. Verger and Jain et al have presented cogent accounts of the kinetics of interfacial catalysis by phospholipases. The complexity of the problem is illustrated in the diagram shown in Fig. 2 showing how the enzyme in the aqueous phase can bind to the interface (designated by the asterisk) and then become activated. Once this is achieved, E catalyzes conversion of S to release P. ... 

There are many enzymes that have a specific binding site Ca in the active center and for which Ca has an essential role in catalysis. An example of a Ca -dependent enzyme is phospholipase A2. Phosphohpase A2 catalyses the hydrolysis of fatty acid esters at the 2 position of phosphohpids (see Fig. 5.24), whereby Ca plays an essential role. The enzyme has two Ca ions boimd tightly at the active center. One of the two Ca ions is directly involved in catalysis. It binds the substrate in the groimd state and also helps to neutralize charge in the transition state of ester hydrolysis. The second Ca ion is assigned a role in stabilization of the transition state, in addition to a structural fimction (White et al., 1990). 

This book provides a complete coverage of phospholipases from the technicalities of assay of phospholipases to the proposed mechanism of catalysis. 

Waszkowycz, B. Hillier, I. H. Gensmantel, N. Payling, D. W. Combined quantum mechanical-molecular mechanical study of catalysis by the enzyme phospholipase A2 an investigation of the potential energy surface for amide hydrolysis, 7. Chem. Soc., Perkin Trans. 2 1991, 2025-2032. 

In summary, this approach is much better suited for the analysis of lipases and phospholipases than the confocal approach described in 2.1 since all steps of the catal3dic cycle can be observed. Interfacial enzymology is a growing held of research [32,45] and the method described here can contribute to a more detailed understanding of catalysis at interfaces. 

Fig. 6. The primary calcium ion of secretory phospholipases A .. Stereoviews showing the coordination chemistry of the primary calcium ion in (A) the absence (Scott etal, 1990a) and (B) the presence of a transition-state analog (White el ai, 1990). Water molecules are labeled W, the proposed attacking nucleophile is inscribed, and tbe primary calcium ion (Ca) is identified. P2 stimulates the tetrahedral intermediate of catalysis and P3 corresponds to the, sn-3 phosphate (copyrighted 1990 by the American Association for the Advancement of Science).

Fig. 7. Class I/II secretory phospholipase Aj catalysis (Scott et al., 1990a). (A) Catalytic attack on productively bound substrate. (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...

Fig. 8. The tetrahedral transition state of secretory phospholipase A2 catalysis. Stereo pairs showing the interaction of the cocrystallized transition-state analog (l-O-octyl-2-heptylphosphonyl-sn-glycero-3-phosphoethanolamine) with the active site of (A) the class I enzyme from Naja naja atra venom (White et al, 1990) and (B) the class 111 enzyme from bee venom (Scott et al., 1990b). The interaction of the transition-state analog with the class II human nonpancreatic enzyme differs only in the types of side chains that interact with the sn-3 substituent [(A) reproduced from ott et al., 1992, copyrighted by The Journal of Biological Chemistry].

Jain, M. K., and Berg, O. G. (1989). The kinetics of interfacial catalysis by phospholipase A2 and regulation of interfacial activation Hopping versus scooting. Biochim. Biophys. Acta 1002, 127-156. 

Jain, M. K., Ranadive, G., Yu, B.-Z., and Verheij, H. M. (1991). Interfacial catalysis by phospholipase A2 Monomeric enzyme is fully catalytically active at the bilayer interface. Biochemistry 30,7330—7340. 

There are many enzymes that have a specific Ca2+-binding site in the active center and for which Ca2+ has an essential role in catalysis. An example of a Ca2+-depen-dent enzyme is phospholipase A2. Phospholipase A2 catalyzes the hydrolysis of fatty acid esters at the 2 position of phospholipids (see Fig. 5.28), where Ca2+ plays... 

Important factors when considering the enhanced hydrolysis at interfaces are the substrate environment in the monolayer and the need to transfer a substrate molecule from this monolayer to the active site. Interfacial disorder may provide an important parameter that facilitates such transfer of substrate to the active site. Phospholipase activity is enhanced under conditions that affect phospholipid fluidity, packing density of the phospholipids, and polymorphism of the aggregate. A highly ordered structure seen with phosphatidylcholine either above or below the transition temperature tends to give low rates of hydrolysis. Discontinuities in such ordered structures occur at temperatures close to the transition temperatures and the presence of other lipids such as anionic lipids or non-bilayer-forming phospholipids promote catalysis by perturbing the interface. 

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