Enzymes sulfhydryl protease

Proteolytic enzymes can be divided into the following four groups the acid proteases, the serine proteases, the sulfhydryl proteases, and the metal-containing proteases. 

Liener, I.E. 1974. The sulfhydryl proteases. In Food related enzymes, ed. J.R. Whitaker. Advances in Chemistry Series 136. Washington, DC American Chemical Society. 

Among the proteolytic enzymes, the plant proteases are the most widely used in the food industry. Most of the plant proteases which have been studied are characterized by a free sulfhydryl group which is essential for their activity. The most important of these so-called sulfhydryl or thiol proteases are papain, ficin, and bromelain. Since the literature on these enzymes has been the subject of several recent reviews (i, 2, 3, 4), major emphasis is placed in this presentation on the use of these enzymes in the food industry. Some of the more recent developments relating to the structure and function of the sulfhydryl proteases are discussed. 

The group of cysteine endopeptidases (also called sulfhydryl proteases or thiol proteases) include the higher plant enzymes papain (EC 3.4.22.2) and ficin (EC 3.4.22.3), but also numerous microbial proteolytic enzymes such as Streptococcus cysteine proteinase (EC 3.4.22.10). The enzymes have a rather broad substrate specificity, and specifically recognise aromatic substituents. The specificity is for the second amino acid from the peptide bond to be cleaved. 

The application of papain in peptide synthesis is well established [23-25]. Papain can be used for fhe preparation of di- and tripepfides in an aqueous medium wifh cosolvent addition (up to 40%) and at high pH to promote synthetic activity. The enzyme is a sulfhydryl protease with no homology to the trypsin or subtilase families of hydrolases. Since the catalytic nucleophile is a cysteine and because thioesters are relatively more prone to aminolysis than oxo-esters, the enzyme could be very attractive for synfhesis. However, unlike the case with the thiol variants of some serine hydrolases, fhe proteolytic activity is still high, and the broad substrate range of proteolysis makes peptide substrate and product hydrolysis more problematic than trypsin or chymotrypsin. Extensive enzyme engineering studies on papain are lacking, probably due to the laborious procedure for isolation of active papain from inclusion bodies formed in E. coli. 

We have chosen to discuss enzyme modification of proteins in terms of changes in various functional properties. Another approach might have been to consider specific substrates for protease action such as meat and milk, legumes and cereals, and the novel sources of food protein such as leaves and microorganisms ( ). Alternatively, the proteases themselves provide categories for discussion, among which are their source (animals, plants, microorganisms), their type (serine-, sulfhydryl-, and metalloenzymes), and their specificity (endo- and exopeptidases, aromatic, aliphatic, or basic residue bond specificity). See Yamamoto (2) for a review of proteolytic enzymes important to functionality. 

Latent forms of MMPs can be activated by mechanisms which cause the dissociation of the intramolecular complex between a particular cysteine residue and the required zinc metal ligand (a complex that blocks the active site) [47], This occurs because the cysteine of the latent enzyme is coordinated to the active site in a particular way that blocks the MMP active site. Collectively, the activation of MMPs occurs through a process which has been termed the cysteine-switch . Activators of the MMPs include proteases (e.g. plasmin), conformational perturbants (SDS, NaSCN), heavy metals and organomercurials (e.g. Au(I) compounds, Hg(II)), oxidants (e.g. OC1-), disulfide compounds (e.g. GSSG) and sulfhydryl alkylating agents (e.g. V-ethylmaleimide) [47 and refs, therein]. 

Pre-slaughter injection of the live animal has proved to be the most effective method of introducing proteolytic enzymes into meat so that they penetrate uniformly into the furthest interstices of the tissue (31). Oxidized papain (103) and proteases whereby the sulfhydryl group in the active site is reversibly blocked (104) have been injected into the jugular veins of animals approximately 30 min before slaughter to distribute the enzyme in the tissues. Subsequently, in the reducing environment of the meat, the protected enzymes are activated. However, the enzymes are apparently active only between 50°C and 82°C or while the meat is cooking. 

Actinidin is a cysteine protease that requires a free sulfhydryl group for activity (Arcus, 1959 McDowall, 1970). This protease is composed of 220 amino acid residues with molecular mass of 23,500 (Came and Moore, 1978). The amino acid sequence of the enzyme shows considerable homology with papain, a well-known cysteine protease in immature papaya fruit (Came and... 

Cathepsins. Cathepsins are intracellular proteases of animal origin. The occurrence of several such enzymes has been demonstrated in various tissues, including spleen, pituitary gland, kidney, thymus, etc. It is obvious that there is no reason to anticipate that all cathepsins will have similar properties to each other or to any other proteases. Cathepsins have been designated by both Roman numerals and by letters. Some of these enzymes have been identified with enzymes purified independently, as cathepsin III with leucine aminopeptidase. Several are activated by sulfhydryl compounds, some by metals. The isolation of the various cathepsins and studies of their substrate specificities are subjects currently under investigation, but because of the lower concentration of enzyme in the source materials and the number of related enzymes present, this area of investigation has not reached the development of the study of digestive enzymes. 

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