Water interface, phospholipase

An anisotropy decay more typical of proteins is shown by phospholipase A2. This enzyme catalyzes the hydrolysis of phospholipids and is active mostly when located at a lipid-water interface. Phospholipase Ai has a single tryptophan resichie (trp-3), which serves as the intrinsic probe. The anisotropy decay is clearly more complex than a single exponential. At long times, the correlation time is 6.5 ns, consistent with overall rotational diffusion. However, in comparison with LADH, there is a dramatic decrease in anisotropy at short times (Figure 11.14), The correlation time of the fast component is less than 50 ps, and this motion accounts for one-(hird of the total anisotropy. Independent tryptophan motions have been observed in a large number of proteins " and have been predicted by molecular dynamics calculations. Fast components in the anisotropy decay are also observed for labeled pro teins. Hence, segmental motions of intrinsic and extrinsic fluorophores appear to be a common feature of proteins. 

The lipases and phospholipase Aj differ from classic esterases in that their natural substrates are insoluble in water and their activity is maximal only when the enzyme is adsorbed to the oif/water interface. Therefore a special treatment of the enzyme kinetics of these enzymes is imposed. The term substrate concentre thm becomes different, as only the substrate present in the interface is available for the enzyme. Consequently, the interface itself becomes the substrate. While in homogeneous svsims. the enzymarlr wngk snare is in three dimensions- and... 

Jain, M. K., Rogers, J., and de Haas, G. H. (1988). Kinetics of binding of phospholipase A2 to lipid/water interfaces and its relationship to interfacial activation. Biochim. Biophys. Acto 940,51-62. 

Pieterson, W. A., Vidal, J. C., Volwerk, J.J., and de Haas, G. H. (1974b). Zymogen-catalyzed hydrolysis of monomeric substrates and the presence of a recognition site for lipid-water interfaces to phospholipase As. Biochemistry IS, 1455—1460. 

Volwerk, J. J., Jost, P. C., de Haas, G. H., and Griffith, O. H. (1986). Activation of porcine pancreatic phospholipase A2 by the presence of negative charges at the lipid-water interface. Biochemistry 25, 1726-1733. 

Pancreatic lipase, in the presence of bile salts and coUpase, acts at the oil-water interface of the triglyceride emulsion to produce fatty acids and 2-monoacylglycerols. Cohpase is secreted in pancreatic juice as an inactive proenzyme, which is converted to the active form by trypsin. Other significant enzymes involved in the breakdown of fats within the intestinal lumen are cholesterol ester hydrolase, phospholipase A, and a nonspecific bile salt-activated lipase. 

Phospholipase A2 (EC 3.1.1.4) " " is a member of a class of lypolytic enzymes that hydrolyze their lipid substrates at an organized lipid-water interface. This enzyme specifically catalyses the hydrolysis of the 2-acyl ester bond of 3-5 -phyosphoglycerides. It has an absolute requirement for Ca " and binds this ion in a 1 1 molar ratio to the enzyme, with a dissociation constant of 2-4 mM. The x-ray structure of the 124-residue bovine enzyme has been determined. It has about 50% a-helical and 10% j8-sheet structure. Ca " " is bound at the active site (Figure 3) and is coordinated to backbone carbonyl atoms of Tyr-28, Gly-30, Gly-32, the two carboxylate oxygens of Asp-49 and two HjO molecules, for a total coordination number of seven. As was the case for staphylococcal nuclease, the Ca " " ligands are supplied from noncontiguous regions of the polypeptide chain. 

This interpretation is supported by the different mechanism of apoHBD release by phospholipase A in the successive steps of purification of the apoHBD (Vidal et al., 1976). In fact, solubilization of apoHBD from the mitochondrial membrane require phospholipid hydrolysis, while apoHBD release from the enzyme-lecithin complex is achieved by competitive binding of phospholipase to the lecithin-water interface without phospholipid hydrolysis. 

The esterases are involved in the hydrolysis of ester linkages of various types. The products formed are acid and alcohol. These enzymes may hydrolyze triglycerides and include several lipases for instance, phospholipids are hydrolyzed by phospholipases, and cholesterol esters are hydrolyzed by cholesterol esterase. The carboxylesterases are enzymes that hydrolyze triglycerides such as tributyrin. They can be distinguished from lipases because they hydrolyze soluble substrates, whereas lipases only act at the water-lipid interfaces of emulsions. Therefore, any condition that results in increased surface area of the water-lipid interface will increase the activity of the enzyme. This is the reason that lipase activity is much greater in homogenized (not pasteurized) milk than in the non-homogenized product. Most of the lipolytic enzymes are specific for either the acid or the alcohol moiety of the substrate, and, in the case of esters of polyhydric alcohols, there may also be a positional specificity. 

Like the lipase, the phospholipase hydrolyzes its substrates at an interface in this case the interface of micelles and water. However pure phospholipid micelles are not digested, and even micelles in which the substrate molecules are spaced apart by inclusion of solvents such as ether or by cationic detergents are not attacked. Micelles containing anionic detergents such as bile salts are attacked. The enzyme requires a negative surface charge on the micelle even though the substrate molecule itself may be an electrically neutral lipid such as phosphatidyl choline. 

For lipolytic enzymes—lipases and phospholipases— Braco and co-workers demonstrated that the presence of surfactants (e.g., octyl P-o-glucopyranosides) and phospholipids in the prelyophilization aqueous phase at concentrations above their critical micellar concentrations (i.e., in the form of micelles or vesicles), increased enzymatic activity by several orders of magnitude. It is believed for lipases that the formation of a small bulk aqueous interface induces an open conformation of the enzyme, where a lid that covers the active site recedes. Treatment of crude lipase with 2-propanol may also promote an open conformation of lipases. Similar to bioimprinting, the activation agent, the surfactant, must be rinsed away from the lyo-philized enzyme by anhydrous solvent prior to use. The activity enhancement decreases with increasing water content in the reaction medium. ... 

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