Pepsin three-dimensional structure

Prabakaran, S., Tepp, W. and DasGupta, B.R., Botulinum neurotoxin types B and E purification, limited proteolysis by endoproteinase Glu-C and pepsin, and comparison of their identified cleaved sites relative to the three-dimensional structure of t q)e A neurotoxin, Toxicon, 39, 1515-1531, 2001. 

Pepsin (EC 3.4.23.1) is a typical aspartic proteinase produced in the gastric mucosa of vertebrates as a zymogen form [10], This enzyme has been extensively characterized, and its three-dimensional structure has been determined at high resolution. Porcine pepsin, in particular, has been studied as model to analyze the structure-function relationship of the aspartic proteinases. Although the aspartic proteinases including mammalian and fungal enzymes are quite similar in their three-dimentional structures, there are drastic differences in the catalytic properties, especially in substrate specificities. 

It is clear that a great deal of work is still required in the field of acid proteases before we reach the level of understanding attained for other groups of proteolytic enzymes. Fortunately the amino acid sequence work on at least three enzymes—pepsin, chymosin, and penicillopepsin— is well advanced, and complete three-dimensional structures should become available in the near future. A tentative structure for rhizopus-pepsin has been obtained, but in the absence of sufficient sequence information interpretation of the electron density maps is difficult. We can... 

This discussion of the metalloexopeptidases has focused on the general role of these enzymes in the conversion of dietary proteins into amino acids. In particular, the apparent synergistic relationship which the pancreatic carboxypeptidases have with the major endopeptidases, trypsin, chymotrypsin, and pepsin, in order to facilitate formation of essential amino acids has been stressed. The chemical characteristics, metalloenzyme nature, and mechanistic details of a representative of each class of exopeptidase have been presented. Leucine aminopeptidase from bovine lens was shown to be subject to an unusual type of metal ion activation which may be representative of a more general situation. Carboxypeptidase A of bovine pancreas was discussed in terms of its three-dimensional structure, the implications of x-ray crystallography to mecha ... 

Aspartic peptidases have so far been described for all endopeptidases. Unfortunately, the tertiary structure has only been elucidated for four families. Endopeptidases of the family A1 consist of two lobes, with the active site between them. One lobe has been derived from the other by gene duplication. In the active site each lobe, with very similar three-dimensional structures, bears one Asp residue of the catalytic dyad. It is interesting to note that the crystal structure of retropepsin from family A2 of clan AA showed a single lobe with one catalytic Asp residue with structural similarity to one lobe of the pepsin from family Al. Retropepsin is only active as a homodimer forming the catalytic site between the two monomeric molecules. There is evidence that the peptidases of families Al and A2 have evolved from a common ancestor. Unfortunately, a number of other families could not yet been assigned to any clan. 

Although the cytoplasm of the cell and the fluids that bathe the cells have a pH that is carefully controlled so that it remains at about pH 7, there are environments within the body in which enzymes must function at a pH far from 7. Protein sequences have evolved that can maintain the proper three-dimensional structure under extreme conditions of pH. For instance, the pH of the stomach is approximately 2 as a result of the secretion of hydrochloric acid by specialized cells of the stomach lining. The proteolytic digestive enzyme pepsin must effectively degrade proteins at this extreme pH. In the case of pepsin the enzyme has evolved in such a way that it can maintain a stable tertiary structure at a pH of 2 and is catalytically most active in the hydrolysis of peptides that have been denatured by very low pH. Thus pepsin has a pH optimum of 2. 

Fig. 16.2. A section of the alignment of sequences of aspartic proteinases achieved by comparing the three-dimensional structures using COMPARER [14]. HIV human immunodeficiency virus RSV Rous sarcoma virus APE endothiapepsin APP penicillopepsin APR rhizopuspepsin PEP hexagonal porcine pepsin CHY calf chymosin. The last letter refers to the amino (N) or car-boxy (C) terminal domains of the pepsins. The coordinates of the three-dimensional structures were obtained from the PDB databank [24]. The amino acid code is the standard one-letter code (see Appendix C) formatted using the following conventions [7] ...

Central to the study of mechanism and specificity of the acid proteinases is a knowledge of the three-dimensional structures of enzymes with different physiological roles. The availability of acid proteinases with either extracellular or intracellular roles in vertebrates as well as similar enzymes from plants, protozoa, and fungi allows a wide range of comparative studies. In our laboratory we have undertaken the x-ray analysis of fungal enzymes, those from Endothia parasitica and Mucor pusillus, and some vertebrate enzymes including chymosin and chicken pepsin, and are beginning work with cathepsin D. 

The evidence from the rotation function that the enzyme from Endothia parasitica is homologous with chymosin and therefore with pepsin is supported by two further arguments. First, a comparison of the three-dimensional structure with that of the enzyme from Rhizopus chinensis shows a striking similarity (see Ref.20 and Chapter 3 in this book) as the first 40 residues of the Rhizopus enzyme are clearly homologous with pepsin, a similar homology for the enzyme from Endothia parasitica is implied. Secondly, the polypeptide chain is of similar length to that of pepsin and visits the centre of the cleft in a way which brings two residues equivalent to Asp-32 and Asp-215 into correlations between the structure of the Endothia parasitica enzyme and pepsin. 

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. 

The pepsin crystals used in the x-ray crystallographic studies are prepared at the presence of ethanol (1,2). It is quite possible that ethanol molecules can eventually be identified on the three-dimensional structure of pepsin crystals to confirm the kinetic studies. 

A number of chemical approaches have been used in the design of renin inhibitors. In the absence of the purified enzyme, most of the early search for inhibitors was carried out using crude renin preparations. The amino acid sequences of mouse, rat and human renin were obtained later on using either the traditional isolation and sequencing techniques or cDNA methodology. Various three-dimensional models of renin were constructed in the early stages, based on the x ray structures of other similar aspartyl proteases, for example endothia-pepsin and penicillopepsin. Later on, the X ray crystal structure of recombinant human renin was reported. The inhibitor design process has been based on some of these models. 

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