Carboxyl protease

The carboxyl proteases are so called because they have two catalytically essential aspartate residues. They were formerly called acid proteases because most of them are active at low pH. The best-known member of the family is pepsin, which has the distinction of being the first enzyme to be named (in 1825, by T. Schwann). Other members are chymosin (rennin) cathepsin D Rhizopus-pepsin (from Rhizopus chinensis) penicillinopepsin (from Penicillium janthinel-lum) the enzyme from Endothia parasitica and renin, which is involved in the regulation of blood pressure. These constitute a homologous family, and all have an Mr of about 35 000. The aspartyl proteases have been thrown into prominence by the discovery of a retroviral subfamily, including one from HIV that is the target of therapy for AIDS. These are homodimers of subunits of about 100 residues.156,157 All the aspartyl proteases contain the two essential aspartyl residues. Their reaction mechanism is the most obscure of all the proteases, and there are no simple chemical models for guidance. 

Aspartate aminotransferase 284, 285 j6-Aspartate decarboxylase 81, 285 Aspartate transcarbamoylase 298, 360 Aspartyl proteases—see carboxyl proteases Aspirin 66, 67 Association rate constants collision theory 54, 158,159 electrostatic enhancement 159-161... 

The naturally occurring inhibitor pepstatin (structure 16.34) binds very tightly to carboxyl proteases Kt with porcine pepsin is 4.5 X 10-11 M.175 The statine residue has a tetrahedral carbon replacing the normal carboxyl carbon, and so... 

Protease Classification. In order to rationally design an inhibitor for a protease it is first necessary to place it into one of four families of proteases (see Table V). For a new enzyme, a study of its inhibition profile with a series of general protease inhibitors is sufficient to classify it into one of the four families. The inhibitors usually used are diiso-propylphosphofluoridate (DFP) or phenylmethane sulfonyl fluoride (PMSF) for serine proteases, 1,10-phenanthroline for metalloproteases, thiol reagents such as iodoacetate or N-ethylmaleimide for thiol proteases, and pepstatin or diazo compounds such as diazoacetyl-norleucine methyl ester for carboxyl proteases. 

Inhibitors of Thiol and Carboxyl Proteases. Thiol proteases are inactivated by peptide chloromethyl ketones (30) and other alkylating agents. Peptide diazomethyl ketones are much more selective reagents since they do not react with serine proteases as do chloromethyl ketones. Diazoketones have been applied to papain and cathepsin B (48) thus far and it appears that they should be applicable to most thiol proteases. Specificity should be obtainable by changing the peptide sequence of the inhibitor to match that of the enzyme being studied. 

No selective irreversible inhibitors for carboxyl proteases have been developed yet. Reagents such as diazoacetyl-DL-norleucine methyl ester and l,2-epoxy-3-(-p-nitrophenoxyl) propane will inhibit many of the carboxyl proteases that have been examined, but little specificity is likely to be observed. 

Rich DH, Bernatowicz MS. Synthesis of analogues of the carboxyl protease inhibitor pepstatin. Effect of structure in subsite P3 on inhibition of pepsin. J. Med. Chem. 1982 25 791-795. 

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

The first hint that two active-site carboxyl groups—one proto-nated and one ionized—might be involved in the catalytic activity of the aspartic proteases came from studies of the pH dependence of enzymatic activity. If an ionizable group in an enzyme active site is essential for activity, a plot of enzyme activity versus pH may look like one of the plots at right. 

As a direct appUcation a potent C2-symmetric HIV-1 protease inhibitor (with two tetrazoles as carboxyl group bioisosteres) was prepared in one pot [77]. The process involved microwave-promoted cyanation followed by conversion of the nitrile group in a tetrazole with azide (Scheme 64). It is notable that the fimctionahzation was achieved so smoothly without side reactions such as the ehmination of water. 

The HIV-1 protease, like other retroviral proteases, is a homodimeric aspartyl protease (see Fig. 1). The active site is formed at the dimer interface, with the two aspartic acids located at the base of the active site. The enzymatic mechanism is thought to be a classic acid-base catalysis involving a water molecule and what is called a push-pull mechanism. The water molecule is thought to transfer a proton to the dyad of the carboxyl groups of the aspartic acids, and then a proton from the dyad is transferred to the peptide bond that is being cleaved. In this mechanism, a tetrahedral intermediate transiently exists, which is nonconvalent and which is mimicked in most of the currently used FDA approved inhibitors. 

Further, Wasserman and coworkers developed a direct acylation of stabilized phosphonium ylides by carboxylic acids in presence of the EDCI/DMAP (way c). This last method allows the introduction of a-aminoacid structures into the resulting P-oxo phosphorus ylides [19-25],opening the way to the total synthesis of depsipeptide elastase inhibitors [22,24] or cyclic peptidic protease inhibitor EurystatinA [20]. 

Lipases are the enzymes for which a number of examples of a promiscuous activity have been reported. Thus, in addition to their original activity comprising hydrolysis of lipids and, generally, catalysis of the hydrolysis or formation of carboxylic esters [107], lipases have been found to catalyze not only the carbon-nitrogen bond hydrolysis/formation (in this case, acting as proteases) but also the carbon-carbon bond-forming reactions. The first example of a lipase-catalyzed Michael addition to 2-(trifluoromethyl)propenoic acid was described as early as in 1986 [108]. Michael addition of secondary amines to acrylonitrile is up to 100-fold faster in the presence of various preparations of the hpase from Candida antariica (CAL-B) than in the absence of a biocatalyst (Scheme 5.20) [109]. 

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

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