Protease specificities, table

Data are adapted from compilations of Dure et at. (1989). O, high osmoticum (PEG or salt) D, desiccation C, cold W, wounding H, heat , untested or unknown. Sd, seed St, stem Pase, protease ASI, a-amylase/subtilisin inhibitor WGA, wheat germ agglutinin RNP, ribonuclear protein not organ specific. Table updated from Skriver Mundy (1990). 

None of the protein inhibitors of NE presented in this chapter are protease specific. They all inhibit more than one protease, but they are protease class specific (Table 5). For example, ai-PI, SLPI, and eglin c are serine-protease inhibitors and inhibit trypsin, chymotrypsin, and cathepsin G in addition to NE. However, ai-PI is an inhibitor of neutrophil PR3, whereas SLPI and eglin c are only very weak inhibitors of PR3 [40]. By contrast, elafin, which shares 38% homology with the C-terminal domain of SLPI, does inhibit PR3. A strong selectivity for NE is important to reduce toxicity resulting from interference of the inhibitor with other proteolytic processes. 

At the center of apoptosis is a family of cysteine proteases, the caspases (cysteine-aspartate proteases) that contain a Cys residue in the catalytic site and cleave their substrates at a consensus (Asp-containing) motif, Asp-Glu-Val-Asp. Caspases display a stringent requirement for Asp in the Pj position of substrates (Nicholson and Thomberry, 1997) and can be classified into three groups based on their substrate specificity (Table 12.8). 

Table 3. Protease specificity. Arrows indicate cleavage site and Xaa represents an amino acid.

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

Table 1.4 I nfluence ofthe organic solvent on the enantioselectivity ofthe protease subtilisin in the kinetic resolution ofthe racemic alcohol (10) (expressed as the enatiomeric ratio E, that is the ratio of the specificity constants of the two enatiomers, (lfcat/ M)s/...

In all the reported examples, the enzyme selectivity was affected by the solvent used, but the stereochemical preference remained the same. However, in some specific cases it was found that it was also possible to invert the hydrolases enantioselectivity. The first report was again from iQibanov s group, which described the transesterification of the model compound (13) with n-propanol. As shown in Table 1.6, the enantiopreference of an Aspergillus oryzae protease shifted from the (l)- to the (D)-enantiomer by moving from acetonitrile to CCI4 [30]. Similar observations on the inversion of enantioselectivity by switching from one solvent to another were later reported by other authors [31]. 

Proteases (endopeptidases or proteinases) commonly used for specific cleavage of proteins are summarised in Table 6.2. Trypsin is almost always used as an enzyme of first choice it is highly specific and stable, has an appropriate pH-optimum and is commercially available in high purity and quality. When the results obtained are ambiguous, or the trypsin cannot be used for any other reason, a different protease can be easily chosen. In all experiments, described here, the trypsin cleavage was applied. 

Table 6.6 Some of the most commonly employed protease inhibitions and the specific classes of proteases they inhibit...

Correlation of Certain Proteases with Metastatic Potential in Model Tumor Systems. A variety of different proteases have been found to correlate with metastatic potential in model tumor systems. Many of these early studies were carried out with B16 mouse melanoma cells. Variants of these cells with different metastatic potential have been selected. In separate experiments, total PA activity, CB activity, and collagenase IV activity have all been found to correlate with metastatic potential in these cells (A4, D6). More recently, levels of mRNA for CB have also been found to correlate with metastasis in these melanoma cells (Ql). Correlations also exist between levels of specific proteases and metastatic ability in a number of other model systems see Table 2 (D6). 

Interpretation of the electron density maps showed that the large subunit could not be modelled beyond His536 (Fig. 6.10), that is fifteen amino acids short of the 551 residues predicted by the nucleotide sequence (Table 6.2). At about the same time, the cleavage of this fifteen-residue stretch, which is performed by a specific protease, was reported to be an obligatory step for the maturation of the enzyme (Menon et al. 1993). It is also of interest to note that in all [NiFe] hydrogenase crystal structures this buried C-terminal histidine is ligated to a metal atom which is either a magnesium or an iron (see above). 

Proteases are the most extensively used enzymes in the food industry, where they act to improve the quality, stability, and solubility of foods. Some of the attributes of enzymes which make them useful in industrial operations include the following 1) They are derived from plants, animal, and microbial sources and are invariably nontoxic substances that are able catalyze specific reactions 2) they are active at very low concentrations under mild conditions of temperature and pH where undesirable side reactions are minimized and 3) they can be inactivated after a desired effect has been achieved. Proteases from plant, animal, and microbial sources find extensive use as food processing aids.(32) Some of the applications of proteases in the food industry are summarized in Table I. 

Table I. Action of Some Highly Specific Proteases...

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