Hydrolytic proteases

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

Husum et al. found that the hydrolytic activities of P-galactosidase from E. coli and the protease subtilisin in a 50 % aqueous solution of the water-miscible ionic liquid [BMIM][Bp4] were comparable to those in 50 % aqueous solutions of ethanol or acetonitrile (Entry 9) [37]. 

Proteases are hydrolytic enzymes with important application in industries, in particular, in detergent and in the food industry. A metagenomic study in which 100 000 plasmid clones were screened for proteolytic activity found one positive done, which was determined to be novel by sequencing analysis [84]. 

While catalysis by aspartic proteases involves the direct hydrolytic attack of water on a peptide bond, catalysis... 

Maintenance of similar zinc proteins in proteases and peptidases for digestion in both cell types and then to hydrolytic management of connective tissue in later eukaryotes. 

Polgar, L. (1987) Structure and Function of Serine Proteases in Hydrolytic Enzymes, Neuberger Brocklehurst (eds.) Elsevier Science Publishers B.V. (Biomedical Division) 159-200. 

Tumor cells express many hydrolytic enzymes, particularly peptidases, some of which are partially specific for certain tumor types, e.g., plasmin, plasminogen activator protease, and cathepsins. A number of prodrug strategies have been developed for the tumor-selective delivery of cytotoxic drugs [45-47], as illustrated below with a few representative examples. 

Most enzymes bind their substrates in a non-covalent manner but, for those that do bind covalently, the intermediate must be less stable than either substrate or product. Many of the enzymes that involve covalent catalysis are hydrolytic enzymes these include proteases, lipases, phosphatases and also acetylcholinesterase. Some of these enzymes possess a serine residue in the active site, which reacts with the substrate to form an acylenzyme intermediate that is attacked by water to complete the hydrolysis (Fignre 3.3). 

Another approach for producing proteases with specific properties which differs from the approach based upon mutagenesis of existing proteases is to synthesize peptides which contain the necessary catalytic and substrate binding groups necess for a particular hydrolytic reaction. One can envision rather unlimited... 

Although the hydrolysis of esters with lipases and esterases represents the most common process to obtain chiral intermediates for the synthesis of pharmaceuticals, proteases and other hydrolytic enzymes such as epoxide hydrolases and nitrilases have also been used for this purpose. We show here a few representative examples of the action of these biocatalysts that have been recently published. 

Trifluoromethyl /1-thioalkyls and /1-amino alcohols are often good reversible inhibitors of esterases and proteases, respectively. Depending on the enzymes (serine or aspartyl enzymes), fluorinated alcohols are often less efficient inhibitors than the corresponding ketones, which act as analogues of the transition state (vide infra). Nevertheless, fluoroalcohols inhibit hydrolytic enzymes with high inhibition constants (Figure 7.25)." ... 

In principle, it is not fair, or course, to approach a problem of prebiotic chemistry by using sophisticated techniques of present-day molecular biology, such as phage display. However in this case the particular research question was not the origin of life, but rather the question given a vast library of random polypeptide chains, what is the folding frequency The criterion utilized for determining whether a protein is folded or not was based on resistance to the hydrolytic power of proteases, with... 

The use of enzymes and whole cells as catalysts in organic chemistry is described. Emphasis is put on the chemical reactions and the importance of providing enantiopure synthons. In particular kinetics of resolution is in focus. Among the topics covered are enzyme classification, structure and mechanism of action of enzymes. Examples are given on the use of hydrolytic enzymes such as esterases, proteases, lipases, epoxide hydrolases, acylases and amidases both in aqueous and low-water media. Reductions and oxidations are treated both using whole cells and pure enzymes. Moreover, use of enzymes in sngar chemistiy and to prodnce amino acids and peptides are discnssed. 

As mentioned in part 2.1.3 hydrolytic enzymes are the most frequently used enzymes in organic chemistry. There are several reasons for this. Firstly, they are easy to ttse because they do not need cofactors like the oxidoreductases. Secondly, there are a large amormt of hydrolytic enzymes available because of their industrial interest. For instance detergent enzymes comprise proteases, celltrlases, amylases and lipases. Even if hydrolytic enzymes catalyse a chemically simple reaction, many important featirres of catalysis are still contained such as chemo-, regio- and stereoselectivity and specificity. 

Starch derived from maize, potatoes, barley, cassava or other somces must be pretreated with hydrolytic enzymes (amylases, amyloglucosidase, proteases), which carry out liquefaction, saccharification and protein hydrolysis, respectively, before it can be fermented by yeasts and other microorganisms into potable or non-potable alcohol. Enzymes can be added in the form of malt (germinated barley) or koji (germinated rice), but this is expensive. Therefore, industrial enzymes have nearly totally replaced malt and koji as enzyme sources, thereby not only improving the economics but also the predictability of the process. 

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