Serine proteases covalent intermediate

Catalysis by enzymes that proceeds via a unique reaction mechanism typically occurs when the transition state intermediate forms a covalent bond with the enzyme (covalent catalysis). The catalytic mechanism of the serine protease chymotrypsin (Figure 7-7) illustrates how an enzyme utilizes covalent catalysis to provide a unique reaction pathway. 

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

The presence of a covalent acyl-enzyme intermediate in the catalytic reaction of the serine proteases made this class of enzymes an attractive candidate for the initial attempt at using subzero temperatures to study an enzymatic mechanism. Elastase was chosen because it is easy to crystallize, diffracts to high resolution, has an active site which is accessible to small molecules diffusing through the crystal lattice, and is stable in high concentrations of cryoprotective solvents. The strategy used in the elastase experiment was to first determine in solution the exact conditions of temperature, organic solvent, and proton activity needed to stabilize an acyl-enzyme intermediate for sufficient time for X-ray data collection, and then to prepare the complex in the preformed, cooled crystal. Solution studies were carried out in the laboratory of Professor A. L. Fink, and were summarized in Section II,A,3. Briefly, it was shown that the chromophoric substrate -carbobenzoxy-L-alanyl-/>-nitrophenyl ester would react with elastase in both solution and in crystals in 70 30 methanol-water at pH 5.2 to form a productive covalent complex. These... 

As discussed above, proteases are peptide bond hydrolases and act as catalysts in this reaction. Consequently, as catalysts they also have the potential to catalyze the reverse reaction, the formation of a peptide bond. Peptide synthesis with proteases can occur via one of two routes either in an equilibrium controlled or a kinetically controlled manner 60). In the kinetically controlled process, the enzyme acts as a transferase. The protease catalyzes the transfer of an acyl group to a nucleophile. This requires an activated substrate preferably in the form of an ester and a protected P carboxyl group. This process occurs through an acyl covalent intermediate. Hence, for kineticmly controlled reactions the eii me must go through an acyl intermediate in its mechanism and thus only serine and cysteine proteases are of use. In equilibrium controlled synthesis, the enzyme serves omy to expedite the rate at which the equilibrium is reached, however, the position of the equilibrium is unaffected by the protease. 

Covalent intermediates in serine protease catalysis have played a significant and historical role in our understanding of enzyme mechanism. During catalysis the Substrate will pass through at least two covalent intermediates. The- tetrahedral adduct is formed before generation of the acyl enzyme. Trapping of covalent acyl enzymes from specific substrates is very difficult since in this substrate type the... 

Figure 8 Irreversible inhibitors of proteases. Serine and cysteine proteases can be acylated by aza-peptides, which release an alcohol, but cannot be deacylated due to the relative unreactivity of the (thio) acyl-enzyme intermediate. Reactive carbons, such as the epoxide of E64, can alkylate the thiol of cysteine proteases. Phosphonate inhibitors form covalent bonds with the active site serine of serine proteases. Phosphonates are specific for serine proteases as a result of the rigid and well-defined oxyanion hole of the protease, which can stabilize the resulting negative charge. Mechanism-based inhibitors make two covalent bonds with their target protease. The cephalosporin above inhibits elastase [23]. After an initial acylation event that opens the p-lactam ring, there are a number of isomerization steps that eventually lead to a Michael addition to His57. Therefore, even if the serine is deacylated, the enzyme is completely inactive.

Many clinically important yff-lactamases are serine proteases that catalyse y5-lactam hydrolysis by a double displacement mechanism involving a covalent acyl-enzyme intermediate. Inhibitors of these enzymes exert their effect by the formation of a stable acyl-enzyme complex. In most cases, this is as a result of changes that take place in the acyl residue after interaction with the enzyme, that is, the inhibitors are mechanism-based. In other cases, the inhibition of yS-lactamases may merely be due to the formation of a relatively stable covalent acyl-enzyme complex without additional alteration [31]. 

Figure 1-7. The catalytic triad in serine proteases. The reactive serine forms an acyl enzyme as a covalent intermediate during the proteolytic cleavage of a peptide bond. During substrate binding a proton is transferred from serine 195 to histidine 57, and the positive charge of the imidazole ring is stabilized by interaction with the carboxylate side chain of aspartic acid 102. The numbering corresponds to the structure of chymotrypsin.

Ynenol lactones are also proposed to inactivate serine proteases irreversibly by alkylation of the active site histidine. Acylation of elastase by ynenol lactones produces an electrophilic allenone intermediate (Fig. 51) which covalently modifies and inactivates the enzyme with a partition ratio of 1.7 (Copp et al., 1987). Direct addition of the allenone carboxylic acid is without effect, demonstrating that the inactivator must be tethered in the active site to allow reaction with the enzyme. Substitution a to the lactone carbonyl is required for loss of activity, whereas the rate of inactivation is decreased by substitution at the acetylene terminus, suggesting that allene formation is slowed or that nucleophilic attack on the allene is hindered. 

The enzyme chymotrypsin provides a good example of the strategies and amino acid side chains used by enzymes to lower the amount of activation energy required. Chymotrypsin is a digestive enzyme released into the intestine that catalyzes the hydrolysis of specific peptide bonds in denatured proteins. It is a member of the serine protease superfamily, enzymes that use a serine in the active site to form a covalent intermediate during proteolysis. In the overall hydrolysis reaction, an OH from water is added to the carbonyl carbon of the peptide bond, and an to the N, tha-eby cleaving the bond (Fig. 8.8). The bond that is cleaved is called the scissile bond. 

Acetylcholinesterase is a serine esterase whose catalytic mechanism is similar to that of the serine proteases. As with chymotrypsin and trypsin, the active site of acetylcholinesterase has serine as part of a Ser-His-Asp catalytic triad. The mechanism -will involve covalent tetrahedral and acyl enzyme intermediates in which the substrate is bonded covalently to the active-site Ser. The reaction starts with nucleophilic attack on... 

In the domain of synthetic inhibitors, chloromethylketone derivatives of specific substrates are potent irreversible covalent inhibitors of the serine proteases, alkylating the active-center histidine at N-2. X-ray crystallographic studies led to the suggestion that, in addition to the above alkylation, there was also nucleophilic attack by the active-center serine hydroxyl to form a hemiketal, which is stereochemically analogous to the tetrahedral intermediate purported to occur during catalysis. 

Alternate substrates are processed by an enzyme s normal catalytic pathway to form a stable covalent enzyme-inhibitor intermediate, such as an acyl-enzyme in the case of serine proteases, where the complex is essentially trapped in a potential energy well. As such, the inhibition is both time dependent and active-site directed. Theoretically, alternate substrates are reversible inhibitors, since the enzyme is essentially unchanged rather, it is suspended at a point within the catalytic process. However, in practical terms, the enzyme-inhibitor complex can be of such stability as to render the inhibition virtually irreversible. 

Chymotrypsin is the most-studied member of the serine protease family of enzymes. The enzyme-catalysed hydrolytic reaction has been shown to occur in at least three kinetically distinguishable steps. The first of these consists of a very fast, diffusion-controlled formation of a non-covalent enzyme-substrate complex, followed by an acylation step. In the latter the acyl group of the substrate is covalently attached to a serine alcohol of the active site with the concomitant release of the amine of an amide substrate, or the alcohol of an ester substrate. In a final deacylation step the acyl-enzyme intermediate is hydrolysed by water, thus regenerating the free enzyme and releasing the carboxylic acid ... 

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