Proteases active-site-specific

Eco is a powerful tool for defining the active sites of serine protease due to the extended substrate-like interaction that it makes with the protease. The three-dimensional structure of a complex with eco has many advantages that a structure of a protease alone or bound to a small molecule inhibitor does not have. Eco can be used to take a molecular impression of the serine protease active site and reveal features that determine substrate preference. These features are used to design specific inhibitors with therapeutic prospects. Often, a small molecule inhibitor is used to define a protease active site cleft, but the resulting structures have particular drawbacks. Typically, a small molecule inhibitor lacks the prime side interac-... 

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. 

Figure 1 Diagram of a protease active site. A protease cieaves a peptide at the scissiie bond, and has a number of specificity subsites, which determine protease specificity. Substrates bind to a protease with their non-prime residues on the N-terminai side of the scissiie bond and their prime-side residues C-terminal to the scissiie bond. The cataiytic residues determine the ciass of protease. Serine, cysteine, and threonine proteases hydroiyze a peptide bond via a covalent acyl-enzyme intermediate, and aspartic, giutamic and metaiioproteases activate a water moiecuie to hydroiyze the peptide bond in a non-covalent manner.

Several protease inhibitors are competitive, and they bind in the protease active site, but also they have secondary binding sites outside the active site, which are critical to inhibition. Exosite binding provides two major benefits 1) It increases the surface area of the interaction, which leads to a greater affinity, and 2) it can provide a greatly increased amount of specificity. 

Transition-state inhibitors stably mimic the transition state of the enzymatic reaction, and thereby interact with the substrate-bin-ding and catalytic machinery of the enzyme in a low-energy conformation. Transition-state analogs are competitive, reversible inhibitors, although some have extremely low Kj s and very slow off-rates. All proteases activate a nucleophile to attack a carbonyl, which leads to the formation of a tetrahedral intermediate that then collapses to form the enzyme products—two peptides. Thus, synthetic small molecules that mimic the tetrahedral intermediate of the protease reaction are attractive transition-state analogs. A classic class of protease transition-state inhibitors uses a boronic acid scaffold (4, 10). Boronic acid adopts a stable tetrahedral conformation in the protease active site that is resistant to nucleophilic attack. Boronic acid inhibitors, which are derivatized with different specificity elements, have been developed against every class of protease... 

Phosphonates (Fig. 8) and sulfonates represent a third class of covalent irreversible inhibitors. These inhibitors adopt a stable tetrahedral geometry and are covalently bound transition-state analogs. They often have a peptide-like specificity element, and the electrophilicity of the leaving groups can be modified to mne the reactivity of the inhibitor. These inhibitors are specific for serine proteases, because the serine protease active site has a well-defined oxyanion hole, which stabilizes the transition-state mimic. 

Structural Insights, Chymotrypsin A Serine Protease. Work with interactive molecular models to learn more about the structural bases of active site specificity and reactivity, and some of the ways in which active site residues can be identified. 

Peptidomimetic approaches are heavily used to build protease inhibitor scaffolds. Selective protease inhibitors are quite straightforward to be obtained because of the substrate variety and specificity of the proteases. However, the concept of privileged scaffolds does not carry far for proteases. The unifying element in protease substrates is the extended beta-strand conformation that allows interactions with four to six subpockets in the protease active site (69). Mimics for this conformation have been developed but they still lack universal applicability for the transfer into clinical application (70). 

Figure 4. Protease mediated hydrolysis of peptide bonds. A) Hydrolytic reaction scheme B) Proteases active site is composed of subsites (S). Each S has an affinity for residues (P). This "lock and Key" mechanism dictates protease specificity.

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

The proteases are secreted as inactive zymogens the active site of the enzyme is masked by a small region of its peptide chain, which is removed by hydrolysis of a specific peptide bond. Pepsinogen is activated to pepsin by gastric acid and by activated pepsin (autocatalysis). In the small intestine, trypsinogen, the precursor of trypsin, is activated by enteropeptidase, which is secreted by the duodenal epithelial cells trypsin can then activate chymotrypsinogen to chymotrypsin, proelas-tase to elastase, procarboxypeptidase to carboxypepti-dase, and proaminopeptidase to aminopeptidase. 

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