Serine proteases acyl-enzyme intermediates

FIGURE 16.26 Acyl-enzyme and amino-enzyme intermediates originally proposed for aspartic proteases were modeled after the acyl-enzyme intermediate of the serine proteases. 

The starting point for much of the work described in this article is the idea that quinone methides (QMs) are the electrophilic species that are generated from ortho-hydro-xybenzyl halides during the relatively selective modification of tryptophan residues in proteins. Therefore, a series of suicide substrates (a subtype of mechanism-based inhibitors) that produce quinone or quinonimine methides (QIMs) have been designed to inhibit enzymes. The concept of mechanism-based inhibitors was very appealing and has been widely applied. The present review will be focused on the inhibition of mammalian serine proteases and bacterial serine (3-lactamases by suicide inhibitors. These very different classes of enzymes have however an analogous step in their catalytic mechanism, the formation of an acyl-enzyme intermediate. Several studies have examined the possible use of quinone or quinonimine methides as the latent... 

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... 

An enzyme reaction intermediate (Enz—O—C(0)R or Enz—S—C(O)R), formed by a carboxyl group transfer (e.g., from a peptide bond or ester) to a hydroxyl or thiol group of an active-site amino acyl residue of the enzyme. Such intermediates are formed in reactions catalyzed by serine proteases transglutaminase, and formylglyci-namide ribonucleotide amidotransferase . Acyl-enzyme intermediates often can be isolated at low temperatures, low pH, or a combination of both. For acyl-seryl derivatives, deacylation at a pH value of 2 is about 10 -fold slower than at the optimal pH. A primary isotope effect can frequently be observed with a C-labeled substrate. If an amide substrate is used, it is possible that a secondary isotope effect may be observed as welF. See also Active Site Titration Serpins (Inhibitory Mechanism)... 

Acyl-enzyme intermediates. Serine proteases are probably the most studied of any group of enzymes.229 Early work was focused on the digestive enzymes. The pseudosubstrate, p-nitrophenyl acetate, reacts with chymotrypsin at pH 4 (far below the optimum pH for hydrolysis) with rapid release of p-nitro-phenol and formation of acetyl derivative of the enzyme. 

The catalytic cycle. Figure 12-11 depicts the generally accepted sequence of reactions for a serine protease. If we consider both the formation and the subsequent hydrolysis of the acyl-enzyme intermediate with appropriate oxyanion intermediates, there are at least seven distinct steps. As indicated in this figure, His 57 not only accepts a proton from the hydroxyl... 

Figure 12-15 Schematic drawing of the active site of a cysteine protease of the papain family with a partial structure of an acyl-enzyme intermediate in green. The thiolate-imidazolium pair of Cys 25 His 159 lies deep in the substrate-binding cleft and bridges an interface between two major structural domains, just as the Ser His pair does in serine proteases (Fig. 12-10). This may facilitate small conformational changes during the catalytic cycle. Asn 175 provides a polarizable acceptor for positive charge, helping to stabilize the preformed ion pair, and allows easy transfer of an imidazolium proton to the product of substrate cleavage. The peptide NH of Cys 25 and the side chain of Gin 19 form an oxyanion hole.

Mechanistic Aspects of /8-Lactamase Inhibition The clinically important /3-lactamases, e.g., the penases, TEM(lll). and cephases(I). are serine proteases that form an acyl enzyme intermediate with /1-lactam substrates and /i-lactam-derived /3-lactamase inhibitors. Mechanistic studies using several /3-lactamase inhibitors have been extensively reviewed and a general inhibition scheme is illustrated in Figure 1. 

As well as complexing the substrate to the active site, many enzymes link covalently with the substrate, or a portion of it, to form an additional intermediate. Such intermediates occur in the action of enzymes as diverse as alkaline phosphatase (phosphoryl enzyme), serine and cysteine proteases (acyl enzymes), glycosidases (acylal enzymes) and aldolases. 

With regard to the use of protease in the synthetic mode, the reaction can be carried out using a kinetic or thermodynamic approach. The kinetic approach requires a serine or cysteine protease that forms an acyl-enzyme intermediate, such as trypsin (E.C. 3.4.21.4), a-chymotrypsin (E.C. 3.4.21.1), subtilisin (E.C. 3.4.21.62), or papain (E.C. 3.4.22.2), and the amino donor substrate must be activated as the ester (Scheme 19.27) or amide (not shown). Here the nucleophile R3-NH2 competes with water to form the peptide bond. Besides amines, other nucleophiles such as alcohols or thiols can be used to compete with water to form new esters or thioesters. Reaction conditions such as pH, temperature, and organic solvent modifiers are manipulated to maximize synthesis. Examples of this approach using carboxypeptidase Y (E.C. 3.4.16.5) from baker s yeast have been described.219... 

Three of the four pancreatic proteases (trypsin, chymotrypsin, and elastase) are called serine proteases because they are all dependent for activity on the side chain of a serine residue in the active site. This serine residue attacks the carbonyl group of the peptide bond to cleave the peptide, giving an acyl-enzyme intermediate (Chap. 8). This ester bond is then hydrolyzed in a second step ... 

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.

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.

More than a third of all known proteolytic enzymes are serine proteases (2). The family name stems from the nucleophilic serine residue within the active site, which attacks the carbonyl moiety of the substrate peptide bond to form an acyl-enzyme intermediate. Nucleophilicity of the catalytic serine is commonly dependent on a catalytic triad of aspartic acid, histidine, and serine—commonly referred to as a charge relay system (3). First observed by Blow over 30 years ago in the structure of chymotrypsin (4), the same combination has been found in four other three-dimensional protein folds that catalyze hydrolysis of peptide bonds. Examples of these folds are observed in trypsin. 

In contrast to the equilibrium-controlled approach which ends with a true equUibrium, in the protease-catalyzed kinetically controlled synthesisf l the product appearing with the highest rate and disappearing with the lowest velocity would accumulate. This approach requires the use of acyl donor esters as carboxy components (Ac-X) and is limited to proteases which rapidly form an acyl-enzyme intermediate (Ac-E). Serine and cysteine proteases are known to catalyze acyl transfer from specific substrates to various nucleophihc amino components via an acyl-enzyme intermediate. In reactions of this type, the protease reacts rapidly with an amino acid or peptide ester, Ac-X, to form a covalent acyl-enzyme intermediate, Ac-E, that reacts, in competition with water, with the amino acid or peptide-derived nucleophile HN to form a new peptide bond (Scheme 3). The partitioning of the acyl-enzyme intermediate between water and the added nucleophile is the rate-limiting step. Under kinetic control, and if k4[HN] k3[H20], the peptide product Ac-N should accumulate. However, the soluble peptide product will be degraded if the reaction is not terminated after the acyl donor ester is consumed. 

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]. 

The proposed mechanism of sPLAg hydrolysis is similar to that reported for the serine proteases (Hunkapillar et ai, 1973 Carter and Wells, 1988) with three major exceptions. The first difference is that SPLA2S lack an acyl enzyme intermediate, because a conserved water molecule hydrogen bonded to the catalytic histidine serves as the source of the nucleophile. In the serine proteases, the hydroxyl of the active site serine is deprotonated to create a nucleophile in the acylation step. Virtually any dispersed potential nucleophile (e.g., water, alcohol, hy-droxylamine) can then subsequently attack the carbonyl of the acyl enzyme during the deacylation step. 

Kinetically controlled syntheses, which are more often studied, can only be carried out by enzymes forming a reactive acyl-enzyme intermediate (serine or cysteine protease. Scheme 5). The reaction starts with weakly activated amino acids (e.g. esters), and the rapidly formed reactive intermediate RCOE is attacked by nucleophiles like amines and water. If k-i and A 4[H2NR ] > < 3[H20], the desired peptide accumulates. Short reaction times, low enzyme concentrations and the danger of secondary hydrolysis of the peptide product are characteristics of these reactions. The optimal pH usually lies above pH 8. 

Serine carbohydrate esterases and transacylases. The commonest reaction mechanism is the standard serine esterase /protease mechanism, demonstrated paradigmally for chymotrypsin, involving an acyl-enzyme intermediate. The enzyme nucleophile is a serine hydroxyl, which is hydrogen bonded the imidazole of a histidine residue, whose other nitrogen is hydrogen bonded to a buried, but ionised, aspartate residue (Figure 6.28),... 

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