Reaction of serine proteases

FIGURE 7.2. Two alternative mechanisms for the catalytic reaction of serine proteases. Route a corresponds to the electrostatic catalysis mechanism while route b corresponds to the double proton transfer (or the charge relay mechanism), gs ts and ti denote ground state, transition state and tetrahedral intermediate, respectively. 

FIGURE 7.3. The force fields for the three resonance structures that describe mechanism a for the catalytic reaction of serine proteases. 

FIGURE 7.4. The energetics of the catalytic reaction of serine proteases in a reference solvent cage. 

Wade Harper, J. Powers, J. C. Reaction of serine proteases with substituted 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-7-amino-4-chloroisocoumarins new reactive mechanism-based inhibitors. Biochemistry 1985, 24, 7200-7213. 

Enzymes Reaction of serine proteases with halomethyl ketones,... 

Fig. B.12.1. Schematic diagram of the mechanism of reaction of serine proteases.

Harper, J.W., Hemmi, K., and Powers, J.C., Reaction of serine proteases with substituted isocoumarins discovery of 3,4-dichloroisocoumarin, a new general mechanism-based serine protease inhibitor. Biochemistry 24, 1831-1841, 1985. 

DCI was developed by James C. Powers and coworkers at Georgia Institute of Technology (Harper, J.W., Hemmi, K., and Powers, J.C., Reaction of serine proteases with substituted isocoumarins discovery of 3,4-dichloroisocoumatin, a new general mechanism-based serine protease inhibitor, Biochemistry 24,1831-1841,1985). This inhibitor is reasonably specific, although side reactions have been described. As with the sulfonyl fluorides and DFR the modification is slowly reversible and enhanced by basic solvent conditions and/or nucleophiles. DCI has been used as a proteosome inhibitor. See Rusbridge, N.M. and... 

Reaction of Serine Proteases with Halomethyl Ketones... 

X-ray crystallographic studies of serine protease complexes with transition-state analogs have shown how chymotrypsin stabilizes the tetrahedral oxyanion transition states (structures (c) and (g) in Figure 16.24) of the protease reaction. The amide nitrogens of Ser and Gly form an oxyanion hole in which the substrate carbonyl oxygen is hydrogen-bonded to the amide N-H groups. 

The actual reaction mechanism is very similar for the different members of the family, but the specificity toward the different side chain, R, differs most strikingly. For example, trypsin cleaves bonds only after positively charged Lys or Arg residues, while chymotrypsin cleaves bonds after large hydrophobic residues. The specificity of serine proteases is usually designated by labeling the residues relative to the peptide bond that is being cleaved, using the notation... 

The elucidation of the X-ray structure of chymotrypsin (Ref. 1) and in a later stage of subtilisin (Ref. 2) revealed an active site with three crucial groups (Fig. 7.1)-the active serine, a neighboring histidine, and a buried aspartic acid. These three residues are frequently called the catalytic triad, and are designated here as Aspc Hisc Serc (where c indicates a catalytic residue). The identification of the location of the active-site groups and intense biochemical studies led to several mechanistic proposals for the action of serine proteases (see, for example, Refs. 1 and 2). However, it appears that without some way of translating the structural information to reaction-potential surfaces it is hard to discriminate between different alternative mechanisms. Thus it is instructive to use the procedure introduced in previous chapters and to examine the feasibility of different... 

Figure 2.2 Key reaction intermediates in the reaction pathway of serine proteases.

Compound 51 was found to be unstable and difficult to purify, as described in the literature [93—95]. Therefore, 51 was not isolated, but was instead converted to the stable pinacol 1-acetamido-l-hexylboronate derivative 52. However, the acylated derivative 52 could not be purified by column chromatography as it was destroyed on silica gel and partially decomposed on alumina. Fortunately, we found that it dissolves in basic aqueous solution (pH > 11) and can then be extracted into diethyl ether when the pH of the aqueous layer is 5—6. Finally, pure 52 was obtained by repeated washing with weak acids and bases. It should be mentioned here that exposure to a strongly acidic solution, which also dissolves compound 51, results in its decomposition. Compared with other routes, the present two-step method involves mild reaction conditions (THF, ambient temperature) and a simple work-up procedure. It should prove very useful in providing an alternative access to a-aminoboronic esters, an important class of inhibitors of serine proteases. 

Does the charge relay mechanism play an important role and, if so, what rate enhancement would such a mechanism provide It appears that it will not be necessary to invoke a charge-relay mechanism to account for the rates of a-chymotrypsin reactions in terms of known chemistry. The presence of aspartic acid in the interior of serine proteases could, of course, have structural rather than mechanistic significance. 

Peptide thioesters (Section 15.1.10) are generally prepared by coupling protected amino acids or peptides with thiols and are used for enzymatic hydrolysis. Peptide dithioesters, used to study the structures of endothiopeptides (Section 15.1.11), may be prepared by the reaction of peptide nitriles with thiols followed by thiolysis (Pinner reaction). Peptide vinyl sulfones (Section 15.1.12), inhibitors of various cysteine proteases, are prepared from N-protected C-terminal aldehydes with sulfonylphosphonates. Peptide nitriles (Section 15.1.13) prepared by dehydration of peptide amides, acylation of a-amino nitriles, or the reaction of Mannich adducts with alkali cyanides, are relatively weak inhibitors of serine proteases. 

Hydrogen bonds appear to be essential in all enzyme-catalyzed reactions, although why they are essential and how they promote function is an open question. In recent years a specific hypothesis for their involvement in catalysis has emerged so-called low-barrier hydrogen bonds (LBHB) have been proposed to lower the transition state energy for many enzymatic reactions, including those of serine protease, citrate... 

The active site of serine proteases is characterized by a catalytic triad of serine, histidine, and aspartate. The mechanism of lipase action can be broken down into (i) adsorption of the lipase to the interface, responsible for the observed interfacial activation (ii) binding of substrate to enzyme (iii) chemical reaction and (iv) release of product(s). 

In another example of combinatorial parallel chemistry, we have recently used the Ugi three-component reactions (Ugi 3-CR) to construct a library of 16,840 protease inhibitors (25). It has been demonstrated previously that the Ugi-3CR reaction provides a useful chemical scaffold for the design of serine protease inhibitors N-substituted 2-substituted-glycine /V-ary 1/alky 1 -amidcs have been identified that are potent factor Xa, factor Vila, or thrombin inhibitors. The three variable substituents of this scaffold, provided by the amine, aldehyde, and isonitrile starting materials, span a favorable pyramidal pharma-cophoric scaffold that can fill the S1, S2, and S3 pockets of the respective protease. This library was screened against five proteases (factor Xa, trypsin, uro-... 

One of the most studied hydrolytic enzymes is chymotrypsin which represents a group of serine proteases. It catalyzes the hydrolysis of peptides to amino acids. The reaction mechanism of the reaction is shown in Figure 2. Two amino acid residues,... 

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