Enzyme classes cysteine proteases

Rhinoviruses, which represent the single major cause of common cold, belong to the family of picornavimses that harbors many medically relevant pathogens. Inhibitors of the 3C protease, a cysteine protease, have shown good antiviral potential. Several classes of compounds were designed based on the known substrate specificity of the enzyme. Mechanism-based, irreversible Michael-acceptors were shown to be both potent inhibitors of the purified enzyme and to have antiviral activity in infected cells. 

Structural analysis of the rhinovirus and the hepatitis A virus 3C proteases (Allaire et al. 1994 Matthews et al. 1994) confirmed earlier predictions that the picomavirus 3C proteases are similar to chymotrypsin-Uke serine proteases in their fold. An important difference is that the serine nucleophile of serine proteases is replaced with a cysteine however, the 3C protease is stracturally distinct from the eukaryotic cysteine protease class of enzymes. 

Cysteine proteases are a class of enzymes that have been widely studied over the years. The overall principles of substrate recognition, catalysis, and inhibition are now reasonably well documented. This enzyme class includes the plant proteases such as papain, actinidin, and bromelain, and several mammalian lysosomal cathe-psins. By far the majority of the literature reports dealing with cysteine proteases describe results obtained with the enzyme papain, because it is considered to be the archetype of this enzyme class. 

The cysteine proteases can be divided into three classes the papain-like, the caspases (and related enzymes), and the picorna viral cysteine proteases. The proposed catalytic mechanism for cysteine protease peptide cleavage is related to the serine protease mechanism but with a cysteine thiol acting as the nucleophile that attacks the scissile peptide bond carbonyl. 

In addition to being an inhibitor of papain-like cysteine proteases, cystatin C has recently been shown be an efficient inhibitor of some of the cysteine proteases of another family of cysteine proteases, called the peptidase family C13, with human legumain as a typical enzyme (C6). Human legumain has, like cathepsin S, been proposed to be involved in the class n MHC presentation of antigens (M3). It has also been shown that the cystatin C inhibitory site for mammalian legumain does not overlap with the cystatin C inhibitory site for papain-like cysteine proteases (Fig. 1) and that the same cystatin C molecule therefore is able to simultaneously inhibit one cysteine protease of each type (A 10). 

The unsaturated residues Dha and Dhb are formed by dehydration of serine and threonine residues, respectively, and the thioether linkages Lan and MeLan are generated by intramolecular Michael-type addition of cysteine thiols to the unsaturated sites (e.g., Fig. 3b). These modifications can be performed by either two separate enzymes (LanB and LanC) in class I lantibiotics or a single bifunctional enzyme (LanM) in class II lantibiotics. Typically, proteolysis of the leader sequence is performed by a dedicated protease, either a LanP serine protease (class I) or the cysteine protease domain of a LanT protein (class II). The lanB genes encode large ( 1000 residues) predominantly hydrophilic dehydratases that may be membrane associated. To date, the dehydratase activity of a LanB protein has not been reconstituted in vitro and little is known about the mechanism of catalysis of this group of enzymes. 

Recent achievements in the development of active-site directed affinity probes for proteases and other enzyme classes provide direct chemical labeling of proteases of interest in the biological system (24-27). These specific activity probes allow joint evaluation of selective protease inhibition concomitant with labeling of relevant protease enzymes for more analyses. Moreover, activity-based probes that selectively label the main protease subclasses—cysteine, serine, metallo, aspartic, and threonine—can provide advantageous chemical approaches for functional protease identification. Activity probe labeling of proteases allows direct identihcation of enzyme proteins by tandem mass spectrometry. Such chemical probes directed to cysteine proteases have been instrumental for identification of the new cathepsin L cysteine protease pathway for neuropeptide biosynthesis, as summarized in this article. 

Some of the serine and cysteine TSA moieties are shown in Fig. 15.29. Selective inhibition between these two classes of protease can be achieved easily. For example, trifluorom-ethylketones (61) and peptidyl boronic acids (62) do not efficiently inhibit cysteine proteases. However, selective inhibition of enzymes within each class can be very difficult. 

Inhibitors of cathepsin K illustrate the principles developed to inhibit this class of enzyme. This enzyme sequence was detected in 1994 by sequencing of human DNA for the human genome project (126).Cathepsin K was found to be inhibited by leupeptin (63) and by compound (64), which surprisingly binds "backwards" to the active site (Fig. 15.30). A hypothesis to develop symmetrical inhibitors of cathepsin K derived from the superposition of both aldehydes on the carbonyl carbon this led to the diamino ketone TSA (65). The diamino ketone moiety seems to work in several classes of cysteine proteases (127). 

For soluble and immobilized bromelain, temperature increase is accompanied by a decrease in residual enzyme activity. A more complex form of denaturation occurs with the immobilized enzyme, which may involve a two-phase process. Immobilization offers more resistance to denaturation at the higher temperature of 60°C where the second phase is prolonged by a factor of three [60]. Differential scanning calorimetry experiments showed that bromelain is an exceptional protease among the cysteine proteases, illustrated by the fact that its thermal denaturation is consistent with an irreversible two-state model [61]. Also, the far UV circular dichroism spectrum of bromelain differs from those of papain and chymopapain and therefore represents a third spectral class within the cysteine proteinase family [62],... 

The benefit of addressing the proteome at the level of distinct enzyme classes, as well as the versatility of ABPP reagents, is highlighted in a third example of comparative ABPP profiling. In this study, carried out by Greenbaum and colleagues, activity-based probes were applied to characterize the functional role of the papain subclass of cysteine proteases in the Plasmodium falciparum life cycle [47]. While cysteine proteases are known to be essential for the... 

There are four major classes of proteases for which the catalytic mechanisms have been defined. These are described according to the active component in the enzyme which assists in the binding of the substrate. These are aspartic acid, cysteine, serine and metalloproteases. There are other proteases but their catalytic mechanisms are unclear. For a review of proteases and their mechanisms and inhibitors see (refs 12 and 13 and also Chapter 5). 

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