Sulfhydryl proteases, protease inhibitors

The most convenient way of categorizing the classes of cathepsin inhibitors is based on the nature of the electrophilic warhead that interacts with the sulfhydryl group of the active site cysteine residue. Since a large portion of the binding energy of a cysteine protease inhibitor comes from the covalent interaction with this thiol, the properties of the resulting molecules are largely derived from the electrophile. In broad terms, these inhibitors can be broken down into ketone and nitrile-based reversible covalent inhibitors, or the more recent non-covalent inhibitors based on an aminoaniline template. 

In some instances, it might be necessary to supplement the 0.32M sucrose solution, pH 7.4, with 1 mAf EDTA, 0.25 mM dithiothreitol (DTT), and/or a cocktail of protease inhibitors. Membrane-bound phospholipases and proteases can be activated during cell disruption. EDTA chelates metal ions (calcium and magnesium) that activate certain phospholipases. DTT is a reducing agent that prevents oxidation of functionally important sulfhydryl groups. Cell disruption may also cause release of proteases from lysosomes. Protease attack on membrane proteins can be prevented by addition of protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF an inhibitor of serine proteases) and/or E-64 (an inhibitor of cysteine proteases), to the sucrose solution... 

Trypsin inhibitors Acid protease inhibitors Sulfhydryl protease inhibitors Metalo-protease inhibitors... 

Inhibitors of well known metallo-, sulfhydi yl and serine proteases block viral proteolysis in infected cells. The use of iodoacetamide (lAM) to stabilize poliovirus and foot-and-mouth virus precursors has been described (36, 52). The preciirsors were subsequently cleaved in vitro to yield normal-sized products (36)1 and polio RHA replicase activity was stabilized in cell-free extracts of lAM-treated cells (55) H-ethyl maleimide inactivates an EMC virus-specific protease in reticulocyte lysates (20). These results could signify that a sulfhydryl-type protease, e.g., cathepsin B, is required for some of the cleavage reactions. However, when such compounds are added to cells, numerous proteins react, and there is substantial inhibition of protein synthesis. Energy metabolism in such cells is depressed, and ATP-reqiiiring proteolytic reactions blocked. 

Figure 1. Sulfhydryl-disulfide-interchange and oxidation pathways between thiols (RSH), protease inhibitors (In-S-S-In), and structural proteins (P-S-S-P). The net result is a new network of disulfide bonds and changed structure of the inhibitors.

The discussion of protease inhibitors illustrates one general mode of inactivation of antinutrients or food toxicants by sulf-hydryl compounds. The sections that follow illustrate a second general approach based on combination of nucleophilic sulfhydryl groups with electrophilic double bonds. 

Sulfhydryl protease inhibitors, for example, are found in the seeds of legumes of the genus Vigna inhibitors of acid proteases occur in potatoes, and inhibitors of metaloproteases occur in potatoes, tomatoes and other crops. 

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