Pancreatic enzymes positional specificity

Topical pancreatic lipase substrates like tributyrin and triolein emulsions are hydrolyzed by carboxylester lipase in the presence of bile salt but slowly—at a rate lower than 3% and 0.5%, respectively, of that observed with the lipase-colipase complex. On the other hand, the positional specificity is not restricted all three sn positions of triglycerides can be split by the carboxylester lipase. While long-chain phospholipids are resistant, short-chain phospholipids are readily attacked by carboxylester lipase [40]. The low substrate specificity of carboxylester lipase makes possible an essential role for this enzyme in the hydrolysis of triglycerides containing certain esterified polyunsaturated fatty acids, such as eicosapentaenoic, arachidonic, or linoleic acids [41], and which may be resistant to attack by pancreas lipase (see p. 190). 

The limitations noted for aminopeptidases are true for carboxypeptidases as well. The pancreatic enzyme carboxypeptidase A shows low rates in the hydrolysis of peptides with a basic amino acid (arginine or lysine) as the C-terminal residue. Carboxypeptidase B is particularly effective when the same basic residues occupy the C-terminal position. The yeast enzyme carboxypeptidase Y is less specific and therefore more generally applicable, but probably still unsuited for the elucidation of a longer sequence. 

If only small amounts of 2-acylglycerols are required then it may be found more convenient to hydrolyse an appropriate triacylglycerol with pancreatic lipase. This enzyme is specific for the 1- and 3- positions and thus yields a 2-acylglycerol product. By keeping the incubation times short (e.g. 5 min), acyl migration is minimized. The 2-acylglycerol can then be purified by a boric acid - TLC system (Jensen and Pitas, 1976). 

The specificity of the acid carboxypeptidase displays the features typical of all pancreatic carboxypeptidases, hydrolysis of the specific substrate R-X-Y between X and Y (R = peptide residue, Z-, Bz-, Ac-). The amino acid in position Y must have a free carboxyl group dipeptides (having free amino group) are not hydrolyzed. The enzyme hydrolyzes most of the a-amino substituted peptides. The carboxypeptidase was inactive on a number of small amides tried at pH 3.0. A peculiarity of its specificity, however, was its inability to hydrolyze the peptide bond of tripeptides tried in the Table 11. 

Fecal elastase 1 concentration can be measured by an ELISA test kit using an antibody specific for the human enzyme pancreatin supplements do not interfere with this pancreatic function test and need not be discontinued. Although measurement of fecal elastase 1 excretion appears to be somewhat more sensitive than fecal chymotrypsin, its specificity and positive predictive value are similarly low, and falsepositive results can be expected in patients with intestinal diseases. Conversely, mild-to-moderate stages of pancreatic exocrine insufficiency cannot be diagnosed reliably. 

Lipases that are in-1,3 specific include those from Mucor miehei, Mucor java-nicus, Aspergillus niger. Pseudomonas fluorescens, Rhizopus delemar, Rhizopus arrhizus, and pancreatic lipase (11, 25-27). These enzymes cleave the fatty acids only from the sn-l and sn-3 positions of the glycerol backbone of TAGs. Thus, with these lipases, TAGs are hydrolyzed to afford FFA, 1,2 (2,3)-DAGs and 2-MAGs. 

The enzyme phospholipase A2 (PLA2, EC 3.1.1.4) is widely distributed among various species in the animal kingdom, notably in the pancreatic tissues of mammals and venoms from snakes and bees It specifically catalyzes the hydrolysis of the acyl-ester bond at the sn-2 position of l,2-diacyl-3-.w-phosphoglycerides in the presence of Ca (Dennis, 1983)... 

Pancreatic lipase is the primary digestive enzyme for the breakdown of triglycerides. It acts on triglycerides to hydrolyze the fatty acyl ester bonds. Lipase is specific for the ester bonds in the V- and 3 -positions to produce free fatty acids and P-monoacylglycerols. Pancreatic lipase is strongly inhibited by bile acids and therefore requires the presence of colipase, a small protein that binds to the lipase and activates it. 

For historical reasons many pharmaceutical enzymes are assayed with physiological or biopolymeric substrates (proteins, polysaccharides, bacteria, oil emulsions), which causes a number of theoretical and practical problems. The interpretation of results is difficult when natural substrates are converted into products that are substrates themselves for the next enzymatic attack. Reaction rates often depend on the position of the scissile bonds in the molecule and the chemical nature of the moieties. Hydrolysis can proceed simultaneously on various bonds at various rates. In proteolysis it is assumed that some products are liberated only after denaturation and that during the reaction course new peptide bonds become accessible for hydrolysis. In these cases the enzymatic mechanisms become exceedingly complex, kinetic parameters are apparent values, and experimental results are strongly influenced by the reaction conditions. Reproducibility problems can occur upon assaying proteinases with a limited specificity for particular casein types. Bromelain and pancreatic proteinase, FEP pharmaceutical enzyme standards, are assayed with a casein substrate. The extent of soluble peptide release is a measure of proteolytic activity. However, due to limited specificity, some proteinases release peptides with a nonrandom aromatic amino acid composition. Contamination of casein preparations with protein and of test enzyme substances with other proteinases biases the assay results. Under these conditions, relative assay methods are indicated. 

The bile-salt-dependent lipase of pancreatic juice has many names such as cholesterol esterase, nonspecific lipase, the most rational being carboxyl ester lipase [27], In the case of water-insoluble substrates this enzyme has an absolute requirement for bile salts specifically having hydroxyl groups in the 3a and la positions [28.29]. The best documented role for this enzyme is to allow the absorption of dietary cholesterol, through hydrolysis of cholesterol esters in the lumen. The enzyme also catalyzes the esterification of cholesterol and a role for it has been proposed in cholesterol absorption [30]. In addition, a wide range of primary and secondary fatty acyl esters including glycerides, vitamin A and E esters are hydrolyzed by this enzyme. 

Merrifieid, J. Am. Chem. Soc. 91, 501 (1969) Denkewalter, Hirschmann et at, Ibid. 502. Series of articles describing the total synthesis of a protein having the full enzymic activity of bovine pancreatic RNase N. Fujii, H. Yajima, J. Chem. Soc. Perkin Trans. I 1981, 789-841. Specifically catalyzes the cleavage of the phosphodiester bond between the 3 and 5 positions of the ribose moieties in RNA with the formation of oligonucleotides terminating in 2, 3 -cyclic phos-... 

Acylation of Phospholipids - The esterification of long-chain, unsaturated fatty acids in the sn-2-position could be explained by different enzyme activities such as specific long-chain acyl-CoA synthetases and CoA-dependent or CoA-independent transacylases.6-11 For example, a long-chain acyl-CoA synthetase has been demonstrated in Isolated platelet membranes that is specific for arachidonate and other long-chain unsaturated fatty acids.6,7 Transfer of arachidonic acid between phospholipids has been observed by the action of CoA-dependent and CoA-independent transacylases. The CoA-dependent transacylases were demonstrated in lymphocytes,8 pancreatic acini9 and liver microsomes.10 A CoA-independent transacylase, recently observed in platelets, catalyzes the synthesis of arachidonoyl-plasmenyl-ethanolamine by acylation of lysoplasmenylethanolamine with arachidonic acid derived from phosphatidylcholine. 1... 

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