Pancreatic enzymes elastase

Some of the pancreatic enzymes in the lumen include pancreatic amylase, pancreatic lipase, elastase, trypsin, a-chymotrypsin, and carboxypeptidase A. For example, the aspirin derivatives aspirin phenylalanine ethyl ester, aspirin phenyllactic ethyl ester, and aspirin phenylalanine amide have been studied as substrates for carboxypeptidase A [67,68], with the phenylalanine ethyl ester derivative proving to be the best substrate. This study indicated that the carboxypeptidase A may serve as a reconversion site for many drug derivatives. 

For example, chymotrypsin cleaves peptides on the C-terminal side of aromatic amino acid residues phenylalanine, tyrosine, and tryptophan, and to a lesser extent some other residues with bulky side-chains, e.g. Leu, Met, Asn, Gin. On the other hand, trypsin cleaves peptides on the C-terminal side of the basic residues arginine and lysine. Elastase usually catalyses hydrolysis of peptide bonds on the C-terminal side of neutral aliphatic amino acids, especially glycine or alanine. These three pancreatic enzymes are about 40% identical in their amino acid sequences, and their catalytic mechanisms are nearly identical. 

The mammalian serine proteases have a common tertiary structure as well as a common function. The enzymes are so called because they have a uniquely reactive serine residue that reacts irreversibly with organophosphates such as diisopropyl fluorophosphate. The major pancreatic enzymes—trypsin, chymotrypsin, and elastase—are kinetically very similar, catalyzing the hydrolysis of peptides... 

This group includes the chymotrypsins, trypsin, elastase, thrombin, and subtilisin. The name of this group of enzymes refers to the seryl residue that is involved in the active site. As a consequence, all of these enzymes are inhibited by diisopropylphosphorofluori-date, which reacts with the hydroxyl group of the seryl residue. They also have an imidazole group as part of the active site and they are all endopeptides. The chymotrypsins, trypsin and elastase, are pancreatic enzymes that carry out their function in the intestinal... 

The pancreas of all mammals so far investigated contain an elastase with similar enzymatic reactions (Lewis et al., 1956 Marrama et al., 1959), but immunological differences have been observed between pancreatic elastases from different species (Moon and Mclvor, 1960). Elastase is secreted in the pancreatic juice as an inactive zymogen, proelastase (Grant and Robbins, 1955 Lamy and Lansing, 1961) which, like other pancreatic enzymes, is activated by trypsin or enterokinase. 

The amino acid sequence of the enzyme is homologous with those of the pancreatic enzymes and has been shown by model building to be compatible with a chymotrypsin-like three-dimensional structure and catalytic site (36). The homology in sequence is particularly well marked around His-57. The enzyme s catalytic properties are virtually indistinguishable from those of pancreatic elastase. 

Metalloprotease inhibitors - also known as metalloproteases or zinc proteases - are proteolytic enzymes of which the activity depends on metal ions, normally bound Zn -. Examples of metalloproteases are the pancreatic enzymes carboxypeptidase A and B, elastase, the well-characterized bacterial enzyme thermolysin and the collagenase family (found in both bacterial and mammalian cells, fibroblast collagenase, neutrophil elastase, gelatinase). 

Total volume of pancreatic juice, amount or concentration of bicarbonate, and activities of pancreatic enzymes are measured in duodenal contents. The enzyme most commonly measured is trypsin, but amylase, lipase, chy-motrypsin, and elastase may also be evaluated. The Lundh test consists of administering a standardized meal consisting of 5% protein, 6% fat, 15% carbohydrate, and 74% nonnutrient fiber. Advantages of the Lundh meal are that it provides a physiological stimulus to the pancreas and is simple to administer. However, administration of the meal prevents determination of the total enzyme and bicarbonate or secretory volume. Moreover, it provides inadequate or no stimu-... 

AH of the other noninvasive tests are based on the reduction in the secretion of pancreatic enzymes with direct measurement of enzymes in feces (chymotrypsin or elastase) or detection of products of their catalytic reactions, after oral administration of synthetic substrates, in urine (NBT-PABA or pancreolauryl test) or in breath ( C-mixed chain triglyceride breath test). 

Pancreatic enzymes a group of at least 12 digestive enzymes, including some of the most investigated of all enzymes Autolysis of the pancreas does not occur, because the proteolytic enzymes, trypsin, chymo-trypsin A and B, elastase and carboxypeptidase A and B, and phospholipase A2 are synthesized and stored in the pancreas as inactive zymogens. The other P. e. require effectors for optimal activity, which are present in the duodenum. Trypsin inhibitors in the pancreatic tissue and secretion afford additional protection against proteolytic destruction by active P.e. With the exception of cholesterol esterase (M, 400,000), the M, of P.e. lie between 13,700 (ribonu-clease) and 50,000 (a-amylase). 

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

Human leukocyte elastase is a protease that degrades elastin and other connective tissue components. It is implicated in the pathogenesis of pulmonary emphysema and other inflammatory diseases such as rheumatoid arthritis and cystic fibrosis. Porcine pancreatic elastase has often been used as a model for HLE. Both enzymes have a small primary binding site Si. 

Vijayalakshmi, J. Meyer, E. F. Kam, C.-M. Powers, J. C. Structural study of porcine pancreatic elastase complexed with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin as a nonreactivable doubly covalent enzyme-inhibitor complex. Biochemistry 1991, 30, 2175-2183. 

This example documents the difficulty of rationalizing the results of in vivo investigations when competitive metabolic reactions are seen. In such cases, simpler in vitro systems may be more informative, as exemplified by the hydrolysis of alkyl phenyl carbonates (phenyl-O-CO-O-alkyl) catalyzed by pig pancreatic elastase (EC 3.4.21.36) [15]. The rate of hydrolysis was monitored by following C02 production as with the carbamates discussed above. Indeed, the enzyme-catalyzed hydrolysis yields the phenyl hemiester of carbonic acid (phenyl-O-COOH), which decomposes rapidly to produce C02 and phenol. With these carbonates, the rate of hydrolysis decreased in the series Bu > i-Bu > Et hexyl, the /-Hu and cyclohexyl derivatives being... 

Unfortunately, the size of the crystallographic problem presented by elastase coupled with the relatively short lifedme of the acyl-enzyme indicated that higher resolution X-ray data would be difficult to obtain without use of much lower temperatures or multidetector techniques to increase the rate of data acquisition. However, it was observed that the acyl-enzyme stability was a consequence of the known kinetic parameters for elastase action on ester substrates. Hydrolysis of esters by the enzyme involves both the formation and breakdown of the covalent intermediate, and even in alcohol-water mixtures at subzero temperatures the rate-limidng step is deacylation. It is this step which is most seriously affected by temperature, allowing the acyl-enzyme to accumulate relatively rapidly at — 55°C but to break down very slowly. Amide substrates display different kinetic behavior the slow step is acylation itself. It was predicted that use of a />-nitrophenyl amid substrate would give the structure of the pre-acyl-enzyme Michaelis complex or even the putadve tetrahedral intermediate (Alber et ai, 1976), but this experiment has not yet been carried out. Instead, over the following 7 years, attention shifted to the smaller enzyme bovine pancreatic ribonuclease A. 

The ultimate objective of an X-ray cryoenzymological study is the mapping of the structures of all kinetically significant species along the reaction pathway. In the case of ribonuclease A this has been largely achieved, as described above. Other enzymatic reactions now await application of the same techniques. Unfortunately, not all crystalline enzymes lend themselves to study by this method. In some cases it may be impossible to find a suitable cryoprotective mother liquor in others, the reaction may occur too rapidly at ordinary temperature. A reaction with Acat of 10 seconds and an activation enthalpy of —6 kcal mol will not be quenched even at — 75°C. The approach we have described in this article can be applied to only a small number of enzymes. Two likely candidates for successors to ribonuclease are the enzymes yeast triosephosphate isomerase and porcine pancreatic elastase. 

One of the goals of synthetic medicinal chemistry is to design potent inilibitors of clinically important proteases. Elastase inhibitors may be useful for treatment of emphysema, pancreatitis, and arthritis,a/b while inhibitors of the angiotensinogen-converting enzyme or of renin (Box 22-D) can help control blood pressure. Inhibition of thrombin, factor Xa, or other blood clotting factors (Fig. 12-17) may prevent blood clots and inhibition of the cytosolic tryptase may provide a new treatment for asthma. Inhibition of the cysteine protease cathepsin K may help combat osteoporosis and inhibition of cysteine proteases of corona viruses may fight the common cold. Cysteine proteases of schistosomes are also targets for protease inhibitors.c... 

The mammalian serine proteases appear to represent a classic case of divergent evolution. All were presumably derived from a common ancestral serine protease.23 Proteins derived from a common ancestor are said to be homologous. Some nonmammalian serine proteases are 20 to 50% identical in sequence with their mammalian counterparts. The crystal structure of the elastase-like protease from Streptomyces griseus has two-thirds of the residues in a conformation similar to those in the mammalian enzymes, despite having only 186 amino acids in its sequence, compared with 245 in a-chymotrypsin. The bacterial enzymes and the pancreatic ones have probably evolved from a common precursor. 

In chapter 8, we mentioned that the pancreas secretes trypsin, chymotrypsin, and elastase as inactive zymogens, which are activated by extracellular proteases. Trypsin is activated when the intestinal enzyme enter-opeptidase cuts off an N-terminal hexapeptide. Trypsin in turn activates chymotrypsin by cutting it at the N-terminal end between Arg 15 and lie 16. This type of change in the covalent structure of an enzyme is termed partial proteoly-sis. Delaying the activation prevents the digestive enzymes from destroying the pancreatic cells in which they are synthesized. 

The electrospray ionization mass spectrometry (ESI-MS) analysis of the incubation between porcine pancreatic elastase (PPE) and the tert-butylammonium salt of clavulanic acid 54 (R1 = R2 = H) at time points of between 3 min and 5h revealed that there were no mass increments relative to PPE. However, when the benzyl derivative 54 (R1 = Bn, R2 = H) was used, a peak was observed at 26187 Da after 3 min, which corresponded to the formation of an initial acyl-enzyme complex. The intensity of the peak decreased significantly and two clear additional peaks at 25 968 and 25 967 Da appeared after 5 min, which corresponded to adducts with mass increments at 77 and 88 Da, respectively. The intensities of both peaks decreased after 60 min and almost disappeared after 5 h. The corresponding />-nitrobenzyl ester 54 (R1 = CH2PhN02, Rz = H) showed similar results except that the formation of adducts appeared slightly faster <2000T5729>. 

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