Pepsin catalyzed reaction

The evidence for involvement of an acyl intermediate comes from the fact that pepsin catalyzes the exchange of between free carboxyl groups of acyl amino acids (products) and water 126, 127). This is fully discussed by Knowles 108) and Fruton 46). Attempts to trap putative acyl enzymes have failed so far. Furthermore, Shkarenkova et al. 128) showed that is rapidly incorporated from H2 into an active site carboxyl group of pepsin in the absence of an acyl amino acid, and that the rate of loss from labelled pepsin is similar to the pepsin-catalyzed rate of exchange with acetylphenylalanine. The 0-exchange experiments, therefore, do not require the formation of an acyl intermediate. With this in mind Knowles proposed a mechanism for pepsin-catalyzed reactions 108) which involves a covalent amino intermediate but not an acyl intermediate. 

While the reaction with porcine pepsin (Figure 2) is qualitatively similar to that with penicillopepsin, there are obvious differences. First, hydrolysis predominated over transpeptidation and second, another trans-peptidation product, tri-leucine, was formed. The putative intermediate Leu-Leu-Tyr has, however, been detected. A detailed discussion of the significance of these experiments will be presented elsewhere. For the purpose of this review suffice it to say that they provide strong evidence for the involvement of an compulsory acyl intermediate in some pepsin and penicillopepsin catalyzed reactions. Hence, mechanistic proposals which do not involve acyl intermediates are insufficient for a complete description of pepsin catalyzed reactions. 

Pepsin-catalyzed reactions involve both an amino and an acyl intermediate as proposed by Bender and Kezdy (131), The order of release would be determined by the nature of the substrate. 

There is also the possibility that both acyl and amino intermediates are required for all the pepsin-catalyzed reactions. Bender and Kezdy (131) suggested that two carboxylic acid groups on the enzyme could reversibly form an anhydride. It could then undergo an exchange reaction with the peptide substrate to form an amino intermediate from... 

Figure 3. Proposed mechanisms for pepsin-catalyzed reactions (see text for details). The orientation of the peptide bond does not imply any particular mechanism of the intermediate formation. The positions of the protonated and unprotonated carboxyl groups in the E-S complex could be reversed, depending on the mechanism of interaction.

We therefore propose that not all pepsin-catalyzed reactions proceed via the same mechanism. We suggest that the nature of the substrate will determine the type of covalent intermediate formed on the pathway of the reaction. The proposed pathways are summarized in Figure 3. The first step would, of course, be the formation of an enzyme substrate complex (Reaction I). Reaction II presents the alternative formation of an acyl intermediate or an amino intermediate, depending on the substrate. 

As is true of all proteases, pepsin catalvises the hydrolysis of peptide bonds. Pepsin lends to recognize a specific family of peptide bonds, namely those occurring between lipophilic amino adds. These amino acids, which frequently occupy the interior or core of proteins, are exposed under the denaturing conditions of the stomach. Tl ie products of the pepsin-catalyzed reactions generally are partially digested proteins and polypeptides rather than free amino acids-... 

Pepsin, a peptidase that hydrolyzes proteins, functions in the stomach at an optimum pH of 1.5 to 2.0. How is the rate of a pepsin-catalyzed reaction affected by each of the following conditions ... 

It is clear that this mechanistic interpretation is not unique, because there is no direct evidence for the proposed intermediates. For example, alternative possible explanations are (1) The amino group in the aminoenzyme may not be covalently bound, but may merely be activated by the enzyme and (2) similarly, the acyl transfer reaction of equation 16.31 could occur by the direct attack of Leu-Tyr-Leu on the enzyme-bound Leu-Tyr-Leu. However, M. S. Silver and S, L. T. James169,170 have proposed a further interpretation, based on the observation that small peptides stimulate the pepsin-catalyzed hydrolysis of other peptides by being first synthesized into larger peptides in a condensation reaction that is the reverse of the hydrolytic step e.g., equations 16.33. The idea of the condensation of two small peptides to give a larger peptide at a rate that is relatively fast compared with hydrolysis of the small peptides is quite reasonable... 

However, since the time these mechanisms were proposed, we have obtained more direct evidence for an acyl intermediate in pepsin- and penicillopepsin-catalyzed reactions from transpeptidation reactions which only proceed via an acyl transfer 129). Some of the experimental evi-... 

Six classes of enzyme-catalyzed reaction are recognized in systematic nomenclature [61]. Their names and the type of chemical reaction catalyzed by each are indicated in Table 4.1. All enzymes have systematic names based on the above, but many are known by historically important trivial names e.g. trypsin, chymo-trypsin, pepsin, lysozyme, catalase. 

The rate of enzyme-catalyzed reactions typically shows a marked dependence on pH (Figure 8-7). Many of the enzymes in blood plasma show maximum activity in vitro in the pH range from 7 to 8. However, activity has been observed at pH values as low as 1.5 (pepsin) and as high as 10.5 (ALP). The optimal pH for a given forward reaction may be different from the optimal pH found for the corresponding reverse reaction. The form of tlie pH-dependence curve is a result of a number of separate effects including the ionization of the substrate and the extent of dissociation of certain key amino acid side chains in the protein molecule, both at the active center and elsewhere in the molecule. Both pH and ionic environment will also have an effect on the three-dimensional conformation of the protein and... 

It is suggested that the process being investigated was the intramolecular, self-catalyzed activation of pepsinogen, since (A) the pepsinogen samples used in this study were all prepared in such a way that they could not contain active pepsin (denatured at pH 7 and above (B) activation was carried out at pH 2.5 (and 4.1, and the same result was obtained for the intermolecular catalyzed reaction) and (C) no active protein could ever be detected, except when the molar ratio of pepstatin/pepsinogen was less than 1 1. 

Fahmey and Reid (11) discovered that pepsin catalyzes the hydrolysis of sulfite esters. Extensive kinetic studies on these reactions have been performed in our laboratory (12-15). The following observations suggest that the active sites involved in the hydrolysis of sulfite esters and in the hydrolysis of peptides may be identical, or may at least overlap ... 

The presence of anhydride intermediates during the course of the hydrolysis of sulfite esters catalyzed by pepsin was proposed by May and Kaiser (14). Studies of the catalysis of sulfite ester hydrolysis by model carboxylate species indicated that the presence of anhydride intermediates could be detected in such reactions by the use of nucleophilic trapping reagents (17). Based on the results of the model studies, we were encouraged to attempt to trap the hypothetical anhydride intermediates formed in the pepsin-catalyzed hydrolysis of a sulfite ester using hydroxylamine as the trapping agent, which could lead to the identification of the active sites involved in this reaction. 

The pepsin-catalyzed transpeptidation reactions, both the amino transfer (14) and the acyl transfer types (15) (Fig.6), suggest the formation of at least two intermediates in which fragments of the substrate are bound to the enzyme. For a long time, however, no transpeptidation reaction of the amino transfer type could be found for substrates containing a blocked COOH-terminal carboxyl group adjacent to the bond being cleaved. The existence of the amino-enzyme in the pepsin hydrolysis pathway of conventional substrates was therefore somewhat doubtful (16). As for the transpeptidation of the acyl transfer type, it has been found so far only on substrates with a free NH2-terminal amino group. 

Figure 9. Possible means of H2 °0 incorporation into the product of pepsin-catalyzed transpeptidation reaction of the acyl transfer type. Theoretical values of °0 content in mg per 100 mg sample of (I) in (a) 0 (b) 0 t 5.2 ...

Enzyme Nomenclature. The number of enzymes known exceeds two thousand. A system of classification and nomenclature is required to identify them unambiguously. During the nineteenth century, it was the practice to identify enzymes by adding the suffix -in to the name of their source. Names such as papain, ftcin, trypsin, pepsin, etc, are still in use. However, this system does not give any indication of the nature of the reaction catalyzed by the enzyme or the type of substrate involved. 

Enzymes are named by a systematic set of rules that nobody follows. The only given is that enzyme names end in -ase and may have something in them that may say something about the type of reaction they catalyze—such as chymotrypsin, pepsin, and enterokinase (all proteases). 

M. S. Silver, S. L. T. James, Surprising Consequence of the Tendency of Pepsin to Catalyze Condensation Reaction of Small Peptides , Biochim. Biophys. Acta 1983, 743, 13-22. 

The problem to be solved with respect to the chemical reactions that constitute metabolism and sustain life is that, without the action of catalysts, they are far too slow. Let s consider the digestion of the proteins themselves, an important constituent of our diet. In an enviromnent similar to that of our digestive system, several tens of thousand years would be required to digest half of the protein content of a typical meal in the absence of a catalyst. Clearly, this will not do. In reality, the stomach secretes one protein catalyst, the enzyme pepsin, and the pancreas secretes several enzymes that catalyze the digestion of proteins. In the presence of these enzymes, dietary proteins are fully digested and reduced to their basic constituents, the amino acids, in a matter of hours. Obviously, these enzymes are enormously potent catalysts." ... 

Four reviews dealing with the mechanism of action of pepsin have been published in recent years (46, 73, 108, 117). Other recent publications deal with various aspects of this mechanism 118-120). In this section, therefore, the main emphasis will be placed on the significance of studies on hitherto unobserved pepsin- and penicillopepsin-catalyzed transpeptidation reactions, especially as they aflFect the mechanisms proposed by various authors. The question we are concerned with is the role of the two carboxyl groups which are involved in the catalytic action. We shall not further consider the role of other functional groups which have been discussed in the previous section. 

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