Serine protease catalytic mechanisms

The considerations presented above were based on the specific assumption that the catalytic reaction of the serine proteases involves mechanism a of Fig. 7.2. However, one can argue that the relevant mechanism is mechanism b (the so-called charge-relay mechanism ). In principle the proper procedure, in case of uncertainty about the actual mechanism, is to perform the calculations for the different alternative mechanisms and to find out which of the calculated activation barriers reproduces the observed one. This procedure, however, can be used with confidence only if the calculations are sufficiently reliable. Fortunately, in many cases one can judge the feasibility of different mechanisms without fully quantitative calculations by a simple conceptual consideration based on the EVB philosophy. To see this point let us consider the feasibility of the charge-relay mechanism (mechanism b) as an alternative to mechanism a. Starting from Fig. 7.2 we note that the energetics of route b can be obtained from the difference between the activation barriers of route b and route a by... 

Especially for enzymes, common mechanisms and associated active sites are often observed. This allows searching for appropriate 3D patterns in a database of structures and for checking models to contain such a pattern (for a well known example, see SER-HIS-ASP serine protease catalytic triad [262]). 

Engineering Substrate Specificity. Although the serine proteases use a common catalytic mechanism, the enzymes have a wide variety of substrate specificities. For example, the natural variant subtiHsins of B. amyloliquefaciens (subtiHsin BPN J and B. licheniformis (subtiHsin Carlsberg) possess very similar stmctures and sequences where 86 of 275 amino acids are identical, but have different catalytic efficiencies, toward tetraamino acid -nitroanilide substrates (67). 

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 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. 

Catalysis, specific acid, 163 Catalytic triad, 171,173 Cavity radius, of solute, 48-49 Charge-relay mechanism, see Serine proteases, charge-relay mechanism Charging processes, in solutions, 82, 83 Chemical bonding, 1,14 Chemical bonds, see also Valence bond model... 

Proteases, which originally catalyze the amidic carbon-nitrogen bond breaking, also catalyze ester hydrolysis. However, in this case, the catalytic mechanism is hkely very similar and consists in the preliminary attack of the active site serine on the carbonyl carbon atom [103]. 

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 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... 

SERINE PROTEASES MINIMAL SCHEMES CATALYTIC MECHANISMS... 

Daggett, V., Schroder, S. and Kollman, P. Catalytic pathways of serine proteases classical and quantum mechanical calculations, J. Am. Chem. Soc., 113 (1991), 8926-8935... 

The catalytic mechanism of the subtilisins is the same as that of the digestive enzymes trypsin and chymotrypsin as well as that of enzymes in the blood clotting cascade, reproduction and other mammalian enzymes. The enzymes are known as serine proteases due to the serine residue which is crucial for catalysis (Kraut, 1977 and Polgar, 1987)... 

Whereas standard proteases use serine, cysteine, aspartate, or metals to cleave peptide bonds, the proteasome employs an unusual catalytic mechanism. N-terminal threonine residues are generated by self-removal of short peptide extensions from the active yS-subunits and act as nucleophiles during peptide-bond hydrolysis [23]. Given its unusual catalytic mechanism, it is not surprising that there are highly specific inhibitors of the proteasome. The fungal metabolite lactacystin and the bacterial product epoxomicin covalently modify the active-site threonines and in-... 

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... 

Figure 3.3 (a) Covalent catalysis the catalytic mechanism of a serine protease. The enzyme acetylcholinesterase is chosen to illustrate the mechanism because it is an important enzyme in the nervous system. Catalysis occurs in three stages (i) binding of acetyl choline (ii) release of choline (iii) hydrolysis of acetyl group from the enzyme to produce acetate, (b) Mechanism of inhibition of serine proteases by diisopropylfluorophosphonate. See text for details. 

Carter, P., Abrahmsen, L. and Wells, J.A. (1991) Probing the mechanism and improving the rate of substrate-assisted catalysis in subtihsin BPN. Biochemistry, 30, 6142-6148. Carter, P. and Wells, J.A. (1988) Dissecting the catalytic triad of a serine protease. 

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). 

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