Pepsin catalytic mechanism

Aspartic endopeptidases contain two catalytically essential aspartic residues. Their postulated catalytic mechanism is summarized in Fig. 3.10 with pepsin A (EC 3.4.23.1) as an example [2] ... 

The results above indicated that the mutations had a minimal effect on substrate binding and influenced primarily the properties of enzyme-bound species in the reaction pathway. Additionally, stereochemical analysis of peptide bond hydrolysis (James and Sielecki, 1985) and pepsin crystal stmcture (Sielecki et al., 1990) indicated that glycine 76 is in a position most favorable for interactions with reaction intermediates. A possible involvement of glycine 76 in stabilizing the transition state was suggested as a h5T)othetical catalytic mechanism for pepsin (Pearl, 1987). As indicated above, all the mutants had altered kinetic constants compared to... 

The reaction conditions can be optimized by examining the effect of different factors snch as water content, temperature, pH, surfactant concentration, reaction time, or product yield. Proteases are classified according to their catalytic mechanisms. Four mechanistic classes have been recognized by the International Union of Biochemistry and Molecular Biology serine proteases (chymotrypsin, trypsin, elastase, subtilisin), cysteine proteases (papain, cathepsins, caspases), aspartic proteases (pepsins, cathepsins, lennins), and metallo proteases. 

Penicillopepsin is an acid protease produced by the mold Penicillium janthinellum at pH s less than 4.1 (1). Enzyme production occurs after the mycelial growth has ceased and sporulation has begun (2). The specificity and catalytic mechanism of penicillopepsin are very similar to those of porcine pepsin (3). The two active site aspartic acid residues, Asp-32 and Asp-215, occur in peptide sequences of at least eight amino acid residues which are almost identical in penicillopepsin, pepsin and chymosin (1,4-10). 

Understanding of the detailed catalytic mechanism of porcine pepsin requires knowledge of the three-dimensional structure of its active site. Currently, the results of x-ray crystallographic studies of this enzyme are still preliminary (1,2). The study of specificity and mechanism is, therefore, particularly useful in formulating the interpretation of the catalytic functions of the structural features in the crystallographic models. 

Fruton, J., 1976. The mechanism of die catalytic action of pepsin and related acid proteina.ses. Advances in Enzymology 44 1-36. 

The carboxyl proteases are so called because they have two catalytically essential aspartate residues. They were formerly called acid proteases because most of them are active at low pH. The best-known member of the family is pepsin, which has the distinction of being the first enzyme to be named (in 1825, by T. Schwann). Other members are chymosin (rennin) cathepsin D Rhizopus-pepsin (from Rhizopus chinensis) penicillinopepsin (from Penicillium janthinel-lum) the enzyme from Endothia parasitica and renin, which is involved in the regulation of blood pressure. These constitute a homologous family, and all have an Mr of about 35 000. The aspartyl proteases have been thrown into prominence by the discovery of a retroviral subfamily, including one from HIV that is the target of therapy for AIDS. These are homodimers of subunits of about 100 residues.156,157 All the aspartyl proteases contain the two essential aspartyl residues. Their reaction mechanism is the most obscure of all the proteases, and there are no simple chemical models for guidance. 

Figure 1. Schematic representation of the relationships between proposed catalytic and inhibitory mechanisms. A. Postulated general acid-general base catalyzed mechanism for substrate hydrolysis by an aspartyl protease. The water molecule indicated is extensively hydrogen bonded to both aspartic acid residues plus other sites in the active site (see Reference 16 for details). Hydrogen bonds to water are omitted here. B. Kinetic events associated with the inhibition of pepsin by pepstatin. The pro-S hydroxyl group of statine displaces the enzyme immobilized water molecule shown in Figure lA. Variable aspartyl sequence numbers refer to penicillopepsin (pepsin, Rhizopus pepsin), respectively.

There is also great similarity between aspartic proteinases in terms of interactions with the transition-state analog inhibitors at the catalytic center. The catalytic aspartyl side chains and the inhibitor hydroxyl group are essentially superimposable in both renin complexes. The isostere C-OH bonds lie at identical positions when the structures of inhibitor complexes of several aspartic proteinases are superposed, in spite of the differences in the sequence and secondary structure. Most of the complex array of hydrogen bonds found in endothiapepsin complexes are formed in renin with the exception of that to the threonine or serine at 218, which is replaced by alanine in human renin. The similarity can be extended to all other pepsin-like aspartic proteinases and even to the retroviral proteinases [44,45]. This implies that the recognition of the transition state is conserved in evolution, and the mechanisms of this divergent group of proteinases must be very similar. 

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. 

One of the essential questions relating to the mechanism is whether the catalytic process involves any covalent intermediates between the substrate moieties and the enzyme. In the case of pepsin two possible intermediates could be formed the amino intermediate which involves the transfer of the amino moiety of the substrate to one of the carboxyl groups on the enzyme and the acyl intermediate which involves the transfer of the acyl moiety as shown on page 165. The examples used are substrates involved in transpeptidation reactions in which the respective intermediate has been demonstrated see below). 

Fruton, J.S. (1976) The Mechanism of the Catalytic Action of Pepsin and Related Acid Proteinases, Adv. Enzymol. Relat. Areas Mol. Biol., 44, 1-36. 

Metabolites, in particular enzymes from bacteria and mold, attack the polymer skeleton, but more importantly the additives in the plastic material. Enzymes such as pepsin, trypsin, and chymotrypsin cleave the peptide bond in proteins and polyamides and can also hydrolyze ester bonds [32]. Their catalytic effect can activate hydrogen in the polymer chain, resulting in the formation of free radicals. The results of these processes are destroyed surfaces (Figure 5.371), loss of gloss, and changes in mechanical and electrical properties. 

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