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Acyl-enzyme intermediate, formation

Fig. 3 Lipase-catalyzed reaction pathways of D-lactates (a) and L-lactates (b) acyl-enzyme intermediate formation steps a and e, subsequent dimer ftumation steps i), c, /, and g, and hydrolysis steps d and h. O denotes that the step takes place, whereas x denotes that the step does not take place. In steps b, c, d,f, g, and h, the lipase leaving group is omitted... Fig. 3 Lipase-catalyzed reaction pathways of D-lactates (a) and L-lactates (b) acyl-enzyme intermediate formation steps a and e, subsequent dimer ftumation steps i), c, /, and g, and hydrolysis steps d and h. O denotes that the step takes place, whereas x denotes that the step does not take place. In steps b, c, d,f, g, and h, the lipase leaving group is omitted...
Hydrolysis of esters and amides by enzymes that form acyl enzyme intermediates is similar in mechanism but different in rate-limiting steps. Whereas formation of the acyl enzyme intermediate is a rate-limiting step for amide hydrolysis, it is the deacylation step that determines the rate of ester hydrolysis. This difference allows elimination of the undesirable amidase activity that is responsible for secondary hydrolysis without affecting the rate of synthesis. Addition of an appropriate cosolvent such as acetonitrile, DMF, or dioxane can selectively eliminate undesirable amidase activity (128). [Pg.345]

FIGURE 16.21 Burst kinetics observed iu the chymotrypsiii reaction. A burst of nitrophe-nolate production is followed by a slower, steady-state release. After an initial lag period, acetate release is also observed. This kinetic pattern is consistent with rapid formation of an acyl-enzyme intermediate (and the burst of nitrophenolate). The slower, steady-state release of products corresponds to rate-limiting breakdown of the acyl-enzyme intermediate. [Pg.516]

In the chymotrypsiii mechanism, the nitrophenylacetate combines with the enzyme to form an ES complex. This is followed by a rapid second step in which an acyl-enzyme intermediate is formed, with the acetyl group covalently bound to the very reactive Ser . The nitrophenyl moiety is released as nitrophenolate (Figure 16.22), accounting for the burst of nitrophenolate product. Attack of a water molecule on the acyl-enzyme intermediate yields acetate as the second product in a subsequent, slower step. The enzyme is now free to bind another molecule of nitrophenylacetate, and the nitrophenolate product produced at this point corresponds to the slower, steady-state formation of product in the upper right portion of Figure 16.21. In this mechanism, the release of acetate is the rate-llmitmg step, and accounts for the observation of burst kinetics—the pattern shown in Figure 16.21. [Pg.516]

FIGURE 16.22 Rapid formation of the acyl-enzyme intermediate is followed by slower product release. [Pg.516]

Transition-state stabilization in chymotrypsin also involves the side chains of the substrate. The side chain of the departing amine product forms stronger interactions with the enzyme upon formation of the tetrahedral intermediate. When the tetrahedral intermediate breaks down (Figure 16.24d and e), steric repulsion between the product amine group and the carbonyl group of the acyl-enzyme intermediate leads to departure of the amine product. [Pg.519]

Fig. 8.2 Interaction of transpeptidase (Enz) with its natural substrate, acyl-D-alanyl-D-alanine in the first stage of the transpeptidation reaction to form an acyl-enzyme intermediate. A similar reaction with a penicillin results in the formation of an inactive penicilloyl-enyme complex. Fig. 8.2 Interaction of transpeptidase (Enz) with its natural substrate, acyl-D-alanyl-D-alanine in the first stage of the transpeptidation reaction to form an acyl-enzyme intermediate. A similar reaction with a penicillin results in the formation of an inactive penicilloyl-enyme complex.
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... [Pg.357]

The antibiotic activity of certain (3-lactams depends largely on their interaction with two different groups of bacterial enzymes. (3-Lactams, like the penicillins and cephalosporins, inhibit the DD-peptidases/transpeptidases that are responsible for the final step of bacterial cell wall biosynthesis.63 Unfortunately, they are themselves destroyed by the [3-lactamases,64 which thereby provide much of the resistance to these antibiotics. Class A, C, and D [3-lactamases and DD-peptidases all have a conserved serine residue in the active site whose hydroxyl group is the primary nucleophile that attacks the substrate carbonyl. Catalysis in both cases involves a double-displacement reaction with the transient formation of an acyl-enzyme intermediate. The major distinction between [3-lactamases and their evolutionary parents the DD-peptidase residues is the lifetime of the acyl-enzyme it is short in (3-lactamases and long in the DD-peptidases.65-67... [Pg.373]

A steady-state kinetics study for Hod was pursued to establish the substrate binding pattern and product release, using lH-3-hydroxy-4-oxoquinoline as aromatic substrate. The reaction proceeds via a ternary complex, by an ordered-bi-bi-mechanism, in which the first to bind is the aromatic substrate then the 02 molecule, and the first to leave the enzyme-product complex is CO [359], Another related finding concerns that substrate anaerobically bound to the enzyme Qdo can easily be washed off by ultra-filtration [360] and so, the formation of a covalent acyl-enzyme intermediate seems unlikely in the... [Pg.169]

It is interesting to note that serine peptidases can, under special conditions in vitro, catalyze the reverse reaction, namely the formation of a peptide bond (Fig. 3.4). The overall mechanism of peptide-bond synthesis by peptidases is represented by the reverse sequence f-a in Fig. 3.3. The nucleophilic amino group of an amino acid residue competes with H20 and reacts with the acyl-enzyme intermediate to form a new peptide bond (Steps d-c in Fig. 3.3). This mechanism is not relevant to the in vivo biosynthesis of proteins but has proved useful for preparative peptide synthesis in vitro [17]. An interesting application of the peptidase-catalyzed peptide synthesis is the enzymatic conversion of porcine insulin to human insulin [18][19]. [Pg.69]

Furthermore, the 3D-location of Tyr150 is quite different from that of Glu166 in class-A /3-lactamases. The observed resistance of the methoxylated cephalosporins to class-C /3-lactamases is due to a slow deacylation step [35], This inhibition is the result of the formation of an acyl-enzyme intermediate... [Pg.190]

N-Carbobenzoxy-L-alanine-/>-nitrophenyl ester is a specific substrate for elastase in which the rate-limiting step is deacylation, that is, hydrolysis of the acyl-enzyme intermediate. In 70% methanol over a reasonable temperature range the energy of activation of the turnover reaction, that is, deacylation, is 15.4 kcal mol. In the pH 6-7 region in this cryoprotective solvent, the turnover reacdon can be made negligibly slow at temperatures of -50 C or below. Under such conditions/i-nitro-phenol is released concurrent to acyl enzyme formation in a 1 1 stoichiometry with active enzyme in the presence of excess substrate. In other words, even at low temperatures, the acylation rate is much faster than deacylation and the acyl enzyme will accumulate on the enzyme. The rate of acyl-enzyme formation can be monitored by following the rate of p-nitrophenol release, and thus the concentration of trapped acyl enzyme may be determined. This calculadon has been carried out and... [Pg.256]

Fig. 1 Catalytic mechanism of CALB showing an acylation and deacylation step and the formation of a covalently bound acyl-enzyme intermediate bottom right) [16]... Fig. 1 Catalytic mechanism of CALB showing an acylation and deacylation step and the formation of a covalently bound acyl-enzyme intermediate bottom right) [16]...
In lipase-catalyzed ROP, it is generally accepted that the monomer activation proceeds via the formation of an acyl-enzyme intermediate by reaction of the Ser residue with the lactone, rendering the carbonyl more prone to nucleophilic attack (Fig. 3) [60-64, 94]. Initiation of the polymerization occurs by deacylation of the acyl-enzyme intermediate by an appropriate nucleophile such as water or an alcohol to produce the corresponding co-hydroxycarboxylic acid or ester. Propagation, on the other hand, occurs by deacylation of the acyl-enzyme intermediate by the terminal hydroxyl group of the growing polymer chain to produce a polymer chain that is elongated by one monomer unit. [Pg.60]

Figure 2. Formation of an acyl-enzyme intermediate by peptide bond scission and nucleophilic attack by an amine group to form a new peptide bond (transpeptida-tion), or by water to release the shortened chain (hydrolysis) ( )... Figure 2. Formation of an acyl-enzyme intermediate by peptide bond scission and nucleophilic attack by an amine group to form a new peptide bond (transpeptida-tion), or by water to release the shortened chain (hydrolysis) ( )...
MECHANISM FIGURE 6-21 Hydrolytic cleavage of a peptide bond by chymotrypsin. The reaction has two phases. In the acylation phase (steps to ), formation of a covalent acyl-enzyme intermediate is coupled to cleavage of the peptide bond. In the deacylation phase (steps to ), deacylation regenerates the free enzyme this is essentially the reverse of the acylation phase, with water mirroring, in reverse, the role of the amine component of the substrate. Chymotrypsin Mechanism... [Pg.217]

In addition to participating in acid-base catalysis, some amino acid side chains may enter into covalent bond formation with substrate molecules, a phenomenon that is often referred to as covalent catalysis.174 When basic groups participate this may be called nucleophilic catalysis. Covalent catalysis occurs frequently with enzymes catalyzing nucleophilic displacement reactions and examples will be considered in Chapter 12. They include the formation of an acyl-enzyme intermediate by chymotrypsin (Fig. 12-11). Several of the coenzymes discussed in Chapters 14 and 15 also participate in covalent catalysis. These coenzymes combine with substrates to form reactive intermediate compounds whose structures allow them to be converted rapidly to the final products. [Pg.494]

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. [Pg.610]

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... [Pg.613]

Papain is a protein-hydrolyzing (proteolytic) enzyme with an -SH group and an imidazole group at the active site. Write a reasonable structure for a "tetrahedral intermediate" that would be expected to arise during formation of an acyl enzyme intermediate. [Pg.675]

Steps in the hydrolysis of p-nitrophenyl acetate by chymotrypsin. In the hydrolysis of this and most other esters, the breakdown of the acyl-enzyme intermediate is the rate-determining step. In the hydrolysis of peptides and amides, the rate-determining step usually is the formation of the acyl-enzyme intermediate. This makes the transient formation of the intermediate more difficult to study because the intermediate breaks down as rapidly as it forms. [Pg.163]

The synthetase consists of the three modules E1, E2, and E3 (for a complete description, see Sec. II. A). Each module is composed of an activation site forming the acyl or aminoacyl adenylate, a carrier domain which is posttranslationally modified with 4 -phosphopantetheine (Sp), and a condensation domain (Cl, C2) or, alternatively, a structurally similar epimerization domain (Ep). Activation of aminoadipate (Aad) leads to an acylated enzyme intermediate, in which Aad is attached to the terminal cysteamine of the cofactor (El-Spl-Aad) [reactions (1) and (2)]. Likewise, activation of cysteine (Cys) leads to cysteinylated module 2 [reactions (3) and (4)]. For the condensation reaction to occur between aminoadipate as donor and cysteine as acceptor, both intermediates are thought to react at the condensation site of module 1 (Cl). Each condensation site is composed, in analogy to ribosomal peptide formation, of an aminoacyl and a peptidyl site. In this case of initiation, the thioester of Aad enters the P-site, while the thioester of Cys enters the A-site. Condensation occurs and leaves the dipeptidyl intermediate Aad-Cys at the carrier protein of the second module [reaction (5)]. The third amino acid valine is activated on module 3, and Val is attached to the carrier protein 3 [reactions (6) and (7)]. Formation of the tripeptide occurs at the second condensation site C2, with the dipeptidyl intermediate entering the P-site and the valiny 1-intermediate the A-site [reaction (8)]. [Pg.13]

Efficient modification steps through the proper orientation of the inhibitor reactive group to the enzyme nucleophile is realized by covalent bond formation. A classic example of this type is the modification of a methionine residue of chymotrypsin by /7-nitrophenyl bromoacetyl a-aminoisobutyrate (26)47). In this instance, the reactive group (bromoacetyl) is fixed at the locus near the active site through a covalent bond by means of acyl enzyme intermediates. [Pg.92]


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Acyl intermediate

Acyl-enzyme intermediates

Acylal formation

Acylals formation

Acylation enzymic

Acylic Intermediates

Enzyme acylation

Enzymic formation

Formate intermediates

Formates, acylation

Intermediates formation

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