Proteinase specificity

Sondell, B., Thomell, L.-E., Stigbrand, T., and Egelrud, T., Immunolcoalization of stratum corneum chymotryptic enzyme in human skin and oral epithelium with monoclonal antibodies evidence of a proteinase specifically expressed in keratinizing squamous epithelia, J. Histochem. Cytochem., 42, 459, 1994. 

Broadbent, J.R., Barnes, M., Brennand, C., Strickland, M., Houck, K., Johnson, M.E., Steele, J.L. 2002. Contribution of Lactococcus lactis cell envelope proteinase specificity to peptide accumulation and bitterness in reduced-fat Cheddar cheese. Appl. Enviro. Micobiol. 68, 1778-1785. 

Exterkate, F. A., Alting, A. C., and Bruinenberg, P. G. (1993). Diversity of cell envelop proteinase specificity among strains of Lactococcus lactis and its relationship to charge characteristics of the substrate-binding region. Appl. Environ. Microbiol. 59,3640-3647. 

Figure 11.6 A schematic view of the presumed binding mode of the tetrahedral transition state intermediate for the deacylation step. The four essential features of the serine proteinases are highlighted in yellow the catalytic triad, the oxyanion hole, the specificity pocket, and the unspecific main-chain substrate binding.

A closer examination of these essential residues, including the catalytic triad, reveals that they are all part of the same two loop regions in the two domains (Figure 11.10). The domains are oriented so that the ends of the two barrels that contain the Greek key crossover connection (described in Chapter 5) between p strands 3 and 4 face each other along the active site. The essential residues in the active site are in these two crossover connections and in the adjacent hairpin loops between p strands 5 and 6. Most of these essential residues are conserved between different members of the chymotrypsin superfamily. They are, of course, surrounded by other parts of the polypeptide chains, which provide minor modifications of the active site, specific for each particular serine proteinase. 

The serine proteinases all have the same substrate, namely, polypeptide chains of proteins. However, different members of the family preferentially cleave polypeptide chains at sites adjacent to different amino acid residues. The structural basis for this preference lies in the side chains that line the substrate specificity pocket in the different enzymes. 

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

Serine proteinases such as chymotrypsin and subtilisin catalyze the cleavage of peptide bonds. Four features essential for catalysis are present in the three-dimensional structures of all serine proteinases a catalytic triad, an oxyanion binding site, a substrate specificity pocket, and a nonspecific binding site for polypeptide substrates. These four features, in a very similar arrangement, are present in both chymotrypsin and subtilisin even though they are achieved in the two enzymes in completely different ways by quite different three-dimensional structures. Chymotrypsin is built up from two p-barrel domains, whereas the subtilisin structure is of the a/p type. These two enzymes provide an example of convergent evolution where completely different loop regions, attached to different framework structures, form similar active sites. 

Affinity and specificity of proteinase inhibitors can be optimized by phage display... 

How do the mutations identified by phage display improve binding specificity There is as yet no direct stmctural information on the phage-selected inhibitors however they can be modeled using data from the crystal structures of other Kunitz domains bound to serine proteinases. These studies lead to the conclusion that the mutations identified by phage display improve binding specificity by maximizing complementarity between the... 

Cathepsins are intracellular proteinases that reside within lysosomes or specific intracellular granules. Cathepsins are used to degrade proteins or pqffides that are internalised from the extracellular space. Some cathepsins such as cathepsin-G or cathepsin-K may be released from the cell to degrade specific extracellular matrix proteins. All cathepsins except cathepsin-G (serine) and cathepsin-D (aspartyl) are cysteine proteinases. 

Proteinase-activated Receptors. Figure 1 Activation of proteinase-activated receptors (PARs) through proteolytic cleavage with serine proteinases (1) and independent of cleavage though PAR-specific activating peptides (2). 

The expression of many of these molecules has been studied during various stages of differentiation of normal neutrophils and also of corresponding leukemic cells employing molecular biology techniques (eg, measurements of their specific mRNAs). For the majority, cDNAs have been isolated and sequenced, amino acid sequences deduced, genes have been localized to specific chromosomal locations, and exons and intron sequences have been defined. Some important proteinases of neutrophils are listed in Table 52-12. 

Poly(L-lysine) has also been suggested as a carrier for pepstatin, a specific inhibitor of the lysosomal proteinase cathepsin D, responsible for causing muscle-wasting diseases, such as muscular dystrophy [257],... 

B4. Barrett, A. J., and Starkey, P. M., The interaction of a2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem. J. 133,709-724 (1973). 

---