Serine proteases, covalently bound

Gehlhaar, D. K., Bouzida, D., Rejto, P. A. (1998) Fully automated and rapid flexible docking of inhibitors covalently bound to serine proteases. Proceedings of the 7th International Conference on Evolutionary Programming, MIT Press, Cambridge, MA., pp. 449-461. 

Activation reactions catalyzed by serine proteases (including kallikreins) are an example of limited proteolysis in which the hydrolysis is limited to one or two particular peptide bonds. Hydrolysis of peptide bonds starts with the oxygen atom of the hydroxyl group of the serine residue that attacks the carbonyl carbon atom of the susceptible peptide bond. At the same time, the serine transfers a proton first to the histidine residue of the catalytic triad and then to the nitrogen atom of the susceptible peptide bond, which is then cleaved and released. The other part of the substrate is now covalently bound to the serine by an ester bond. The charge that develops at this stage is partially neutralized by the third (asparate) residue of the catalytic triad. This process is followed by deacylation, in which the histidine draws a... 

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. 

Chymotrypsin is a digestive enzyme and belongs to the family of serine proteases and catalyzes the hydrolysis of proteins and peptides. A part of the substrate is bound covalently to the enzyme. Chymotrypsin hydrolyzes esters as well even if this is physiologically not important. But this fact was of interest for mechanistic studies of chymotrypsin-catalyzed hydrolysis (Figure 23b). The rate-determining step is the hydrolysis of the enzyme-acetyl complex by... 

Chymotripsin (ChT) is a serine protease which was already shown to be inhibited by a specific family of calix[4]arene receptors [26, 31]. P NMR diffusion methods were exploited to probe the molecular interaction of ChT with organo-phosphorus compounds which are known to block the enzyme. In such cases, covalent conjugates with the serine active site were detected on the basis of the relevant decreases of diffusion coefficients of the enzyme bound species, simultaneously enabling the determination of the diffusion coefficient of the enzyme [27]. 

Some speculative, but necessarily incomplete comparison may be made for serine proteases. It is possible to distinguish a binding domain in proteases, the one located toward the C-terminus (C domain), but it is not possible to refer to the other domain as the catalytic one since it contains only two of the three catalytic residues of the charge relay system. Ser 195, which keeps a part of the substrate covalently bound in the acyl-enzyme intermediate pertains to the binding domain. 

In contrast, 5-benzyl-6-chloro-2-pyrone is not a substrate for chymotrypsin, but inactivates the enzyme with the formation of a shoulder in the absorbance spectrum at 320 nm (Westkaemper and Abeles, 1983). The chromophore, which is similar to that of the pyrone ring, appears with kinetics corresponding to the rate of inactivation and is lost on reactivation of the enzyme. The crystal structure and C NMR studies of the modified enzyme demonstrate that the benzyl group is bound in the specificity pocket of the active site and that serine-195 is covalently attached to C-6 of the intact pyrone (Ringe et ai, 1985) (Fig. 49). Inactivation by the 5-benzyI analog is therefore an example of affinity labeling rather than mechanism-based inactivation and emphasizes the importance of the side chain of protease inactivators in proper orientation of the compound in the active site. 

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