Acid proteases specificity

In this paper we discuss the results obtained from an examination of the 2.5 A resolution electron density map, and present some of the data obtained from binding pepstatin (an acid-protease-specific inhibitor) to the enzyme in the crystals. 

BPN is a 275 amino acid protease with a serine in the active site. Since it is functional in an alkaline environment it has potential use in detergent applications. Our program was to change specific characteristics of BPN to make it more effective in certain applications. Two main activities were targeted pH range and oxidative stability (since bleaches are often components of detergents). 

Powers JC, Harley AD, Myers DV. Subsite specificity of porcine pepsin. In Tang J, ed. Acid Proteases-Structure, Function and Biology. New York Plenum Press, 1977 141-157. 

Thermolysin belongs to a class of proteases (called neutral proteases) which are distinct from the serine proteases, sulfhydryl proteases, metal-loexopeptidases, and acid proteases. Neutral proteases A and B from Bacillus subtilis resemble thermolysin in molecular weight, substrate specificity, amino acid content, and metal ion dependence. Since physiological substrates are most likely proteins, it is difficult to design simple experiments that can be interpreted in terms of substrate specificity and relative velocities. Therefore, studies of substrate specificity and other kinetic parameters must be carried out on di- and tripeptides so that details of the mechanism of catalysis can be obtained and interpreted simply. 

All proteolytic enzymes described are fairly non-specific serine endoproteases, cleaving peptide chains preferentially at the carboxyl side of hydrophobic amino acid residues. The enzymes convert their substrates into small, readily soluble fragments which can be removed easily from fabrics. Only serine protease can be used in detergent formulations, as thiol proteases such as papain would be oxidized by the bleaching agents, acidic proteases are not active at common laundry conditions, and metalloproteases such as thermolysin would lose their metal cofactors because of complexation with the water-softening agents or hydroxyl ions. 

Substrates for hepatic metabolism include insulin, glucagon, and t-PAs [89,90]. For insulin, an acidic endopeptidase (termed endosomal acidic insulinase ) appears to mediate internalized insulin proteolysis at a number of sites [91]. Specifically, the endosomal activity results from cathepsin D, an aspartic acid protease [92]. Similarly, proteolysis of glucagon has also been attributed to membrane-bound forms ofcathepsins B and D [93]. 

Acid proteases are inactivated by active-site specific reagents, diazoacetylnorleucine ethyl ester and other diazo compounds, and epoxy (p-nitrophenoxy)propane. Covalently labelled aspartic acid peptides have been isolated from pepsin, chymosin (= rennin), and penicillopepsin. The peptides labelled with the diazo compounds have similar sequences and differ from the epoxy (p-nitrophenoxy)pro-pane labelled peptides. These results indicate two aspartic acids at the active site and suggest homology between the enzymes. The latter is confirmed by a comparison of the sequence data. Studies of the action of porcine pepsin and penicillopepsin on some dipeptides with free N-terminal groups show transpeptidation involving a covalent acyl intermediate. It is proposed that there are differences in the mechanism of action of pepsin which are determined by the nature of the substrate. 

Reaction with Diazo Reagents. The classification of the acid proteases on the basis of their pH optima as suggested by Hartley (43) was useful in the absence of other information on the nature of active site residues. Since Hartley s proposal, however, it has been discovered— first with porcine pepsin—that in the presence of Cu ions these enzymes can be specifically inactivated with diazotized dipeptide esters. An ester linkage is formed between one specific aspartic acid side chain and the inhibitor (50). 

Catalyzed by Acid Proteases Compared with Side Chain Specificity... 

With one recent exception, which is mentioned later, serine protease specificities tend to be related to the nature of the amino acid residue which contributes the carbonyl group to a bond which is susceptible to cleavage, le., to residue Pi of, for example, the polypeptide ... 

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