Water interface, pancreatic lipase

In 1958 Sarda and Desnuelle [79] discovered the lipase activation at the interfaces. They observed that porcine pancreatic lipase in aqueous solution was activated some 10-fold at hydrophobic interfaces which were created by poorly water-soluble substrates. An artificial interface created in the presence of organic solvent can also increase the activity of the lipase. This interfacial activation was hypothesized to be due to a dehydration of the ester substrate at the interface [80], or enzyme conformational change resulting from the adsorption of the lipase onto a hydrophobic interface [42,81,82]. 

Triacylglycerol Upases [EC 3.1.1.3] (also known as triglyceride lipases, tributyrases, or simply as lipases) catalyze the hydrolysis of a triacylglycerol to produce a diac-ylglycerol and a fatty acid anion. The pancreatic enzyme acts only on an ester-water interface the outer ester links in the substrate are the ones which are preferentially... 

In another set of studies, it has been reported that the in vitro digestibility of lipid droplets by pancreatic lipase is significantly affected by emulsifier type (Mun et al, 2006, 2007 Park et al., 2007). Intuitively, one might expect that a thick dense layer of strongly bound protein-polysaccharide complex at the oil-water interface would reduce considerably the in vivo accessibility of lipases, and hence would reduce the rate of human metabolism of fats. Establishment of the validity of this hypothesis must still await consolidation of a substantial body of detailed results from independent systematic studies on a broad range of mixed biopolymer systems. 

Like pancreatic lipase, this enzyme is clearly dependent on a lipid/water interface for maximal activity, where it also may reach a very high catalytic rale, Abo like pancreatic lipase, it is not inhibited by serine esterase inhibitors like DFP orPMSF. 

Figure 6. Hypothetical rhodel of the orientation of pancreatic lipase at the oil-water interface...

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. 

Human pancreatic lipase (HPL) alone is inactive in vitro on an emulsified TAG substrate in the presence of supramiceUar concentrations of bile salts such as those found in the small intestine. Bile salts are amphiphilic molecules that bind to the oil-water interface and prevent pancreatic hpase adsorption, and thus hpo-lysis, from occurring [3, 4]. The inhibition by bile salts can, however, be reversed by the specific pancreatic hpase cofactor cohpase [3, 5-7], via the formation of a specific 1 1 hpase-cohpase complex. 

Momsen, W. E. and Brockmann, H.L. (1976) Effect of Colipase and Taurodeoxy-cholate on the Catalytic and Physical Properties of Pancreatic Lipase B at an Oil-Water Interface. ). Biol. Chem. 251, 378-383... 

G. and Verger, R. (1989) Interface-mediated inactivation of pancreatic lipase by a water-reactive compound 2-sulfo-benzoic cyclic anhydride. Biochemistry 28, 6340-6346... 

Soluble substrates are available which are cleaved by carboxylester lipase in the absence of bile salts, such as the not quite specific para-nitrophenylacetate [40] or the dilaurate ester of fluorescein [51,52] (Fig. 9). However, bile salts not only protect the enzyme, but interact specifically with the enzyme, affecting approximately 7- to 10-fold the activity on water-soluble substrates like para-nitrophenylacetate. When water-insoluble esters like triolein or cholesterol oleate are selected as the substrate, the enzyme is virtually inactive without bile salts, which gives them the character of essential activators [53]. In contrast to pancreatic lipase, carboxylester lipase needs no cofactor like colipase and is not activated by an oilAvater interface. Purified carboxylester lipase loses the majority of its activity by lyophylization but can be stored frozen at -20°C [46]. 

Pancreatic lipase is an enzyme found in intestinal mucosal cells that digests triacylglycerols to a mixture of free fatty acids, glycerol, monoacylglycerols, and diacylglycerols. The enzyme requires calcium and is unusual in catalyzing its reaction at an oil-water interface (Figure 18.6). 

The anomalous activity characteristics have been attributed to conformational changes of the solubilized enzyme [49], but more recent spectroscopic studies seem to indicate that this is not the main cause. Solubilization of an enzyme into microemulsion droplets does not normally lead to major conformational alterations, as indicated, e.g., by fluorescence and phosphorescence spectral investigations [28,50]. The situation is complex, however, and it has been shown by circular dichroism (CD) measurements that the influence of the oil/water interface on enzyme conformation may vary even between enzymes belonging to the same class [51]. In the case of human pancreatic lipase, the conformation of the polypeptide chain is hardly altered after the enzyme is transferred from a bulk aqueous solution to the microenvironment of reverse micelles. Conversely, the CD spectra of the lipases from... 

In the small intestine, pancreozymin causes the gallbladder to contract, and bile, a micellar solution of bile acids, lecithin, and cholesterol, is secreted into the duodenum. Pancreozymin also causes discharge and continued synthesis of pancreatic lipase which adsorbs to the oil-water interface, liberating 2-monoglycerides and fatty acids (76). Whether bile acids adsorb to the interface and if so how they spatially orient with respect to lipolytic products and lipase is unknown. At concentrations below the CMC, bile acids will adsorb to monolayers of lipolytic products (77), but no information is available on the interaction of bile acid solutions above their CMC with monolayers of lipolytic products. Somehow, the lipolytic products are transferred to the bulk phase, where they form mixed micelles with bile acid molecules (Fig. 14). 

The most important and typical enzymes that function at lipid-water interfaces in micelles, liposomes, emulsions, etc., are lipases. Lipases are carboxylic ester hydrolases and have been termed glycerol ester hydrolases (EC3.1.1.3) in the international system of classification. They differ greatly as regards both their origins (bacterial, fungal, plant, mammalian, etc.) and their properties, and they can catalyze the synthesis as well as the hydrolysis of a wide range of different carboxylic esters. Numerous reports have appeared about the structure and function of pancreatic lipases, because they are ubiquitous in mammalian species and play important roles in dietary fat absorption [29,30]. In this part, I will describe a structural feature and its relation to catalytic mechanism at the interfaces of lipases, particularly pancreatic lipases. 

A model for pancreatic lipase has been suggested to account for the enzyme s activity on the oil/water interface (Fig. 3.17). The lipase s hydrophobic head is bound to the oil droplet by hydrophobic interactions, while the enzyme s active site aligns with and binds to the substrate molecule. The active site resembles that of serine proteinase. The splitting of the ester bond occurs with the involvement of Ser, His and Asp residues on the enzyme by a mechanism analogous to that of chymotrypsin (cf. 2.4.2.5). The dissimilarity between pancreatic lipase and serine proteinase is in the active site lipase has a leucine residue within this site in order to establish hydrophobic contact with the lipid substrate and to align it with the activity center. 

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