4-Nitrophenyl acetate reaction mechanism

At the start of this section the cleavage of meta- and para-substituted phenyl acetates by a- and )S-CD was discussed in detail and a variety of evidence was cited that is consistent with the mechanisms A and B, in Scheme 2. Further support for the view that para-substituents tend to force the phenyl group out of the cavity (Scheme 2B) comes from the different effects that neutral additives (potential inhibitors) have on the cleavage of m- and p-nitrophenyl acetate (mNPA and pNPA). In brief, species which bind to CDs, and inhibit the reaction of mNPA, do not necessarily inhibit that of pNPA (Tee and Hoeven, 1989 Tee et al., 1993b). 

The photodecarboxylation of nitrophenyl acetate in aqueous media was also investigated recently89 -92, especially with respect to the kinetic and spectral properties of the photogenerated p-nitrobenzyl carbanion its triplet state (Xmax ca 290 nm) was identified to have a lifetime of 90 nanoseconds at pH > 5.0. The proposed reaction mechanism following 266-nm laser excitation of p-nitrophenyl acetate is summarized in Scheme 792. 

Kirsh et al. 42) prepared apolar derivatives of poly(4-vinylpyridine) by benzylation. With nitrophenyl acetate as the substrate the benzylated catalyst is 100 times more effective than 4-ethylpyridine. A double-displacement mechanism was observed. The rate constants for deacylation of the acylpoly(vinylpyridine) derivatives were about 4 x 10" /sec. The comparable value for a-chymotrypsin is 8 x 10 /sec. The factor of 20 seems small, but it should be kept in mind that deacetylation of a-chymotrypsin is very slow compared with the deacylation reactions involving the natural substrates of the enzyme. 

In 1952, Hartley and Kilby showed that p-nitrophenyl acetate reacts with chymotrypsin, and advanced a two-step mechanism for the process (Hartley and Kilby, 1952). Two years later Hartley showed that a burst of p-nitrophenol was produced in the reaction (Hartley and Kilby, 1954). That is to say, a graph of the production of p-nitrophenol from the chymotryptic hydrolysis of p-nitrophenyl acetate does not seem to begin at the origin, but instead a small amount of p-nitrophenol is produced very rapidly. Fur-... 

Stopped flow kinetics (Gutfreund and Sturtevant, 1956) strongly supported the two-step mechanism for the hydrolysis of p-nitrophenyl acetate previously advanced from Hartley s laboratory. The reaction proceeds by the acetylation of the enzyme at the active site, followed by slower hydrolysis of the resulting acetylchymotrypsin (Scheme 5). This, of course, regenerates the enzyme for further rapid reaction with the substrate. 

FIG. 4. An overall reaction mechanism for 4-nitrophenyl acetate hydrolysis catalyzed... 

These principles appear to hold for nucleophilic catalysis of hydrolysis by other species also. The evidence for catalysis by amino-compounds is discussed below. Catalysis by a wide variety of oxyanions (and other anions) has been measured by several authors, particularly of the hydrolysis of p-nitrophenyl acetate. This is a convenient substrate kinetically, since the release of / -nitrophenoxide is easily followed spectrophotometrically at 400 nm, but perhaps not ideal mechanistically, since, as described above, at least some of its reactions involve a mixture of mechanisms. A selection of data, obtained under the same conditions in one laboratory283, is given in Tables 37 and 38. Some of these data are plotted logarithmically (in Fig. 17) against the... 

Substrate-catalyst interaction is also essential for micellar catalysis, the principles of which have long been established and consistently described in detail [63-66]. The main feature of micellar catalysis is the ability of reacting species to concentrate inside micelles, which leads to a considerable acceleration of the reaction. The same principle may apply for polymer systems. An interesting way to concentrate the substrate inside polymer catalysts is the use of cross-linked amphiphilic polymer latexes [67-69]. Liu et al. [67] synthesized a histidine-containing resin which was active in hydrolysis of p-nitrophenyl acetate (NPA). The kinetics curve of NPA decomposition in the presence of the resin was of Michaelis-Menten type, indicating that the catalytic act was accompanied by sorption of the substrate. However, no discussion of the possible sorption mechanisms (i.e., sorption by the interfaces or by the core of the resin beads) was presented. 

Finally, an aspartic acid residue is necessary for full catalysis and this residue is thought to use its CO2 group as a general base. A chemical model shows that the hydrolysis of f> nitrophenyl acetate in aqueous acetonitrile containing sodium benzoate and imidazole follows the rate law rate — <[p-nitrophenyl acetate] [benzoate] [imidazole]. Suggest a mechanism for the chemical reaction. 

Effective Charge Map for a Putative Stepwise Process. Effective charge maps can also be employed to discount a stepwise process if estimates of the effective charge change from reactant and product to putative intermediates are not consistent with expectation. Consider the Bronsted dependence for reaction of substituted phenolate ions with 4-nitrophenyl acetate (Figure 6). The value of Pnu is approximately 0.80 for attack of substituted phenolate ions on 4-nitrophenyl acetate when the second step of the putative two-step mechanism (decomposition of the putative tetrahedral intermediate) would be rate limiting (pA pA a ) (Scheme 11). 

The catalytic activity of the zinc complexes given in Table 1 for 4-nitrophenylac-etate hydrolysis is well below that found for free OH- or for the zinc center for human CA-II.108 In the case of OH-, this is consistent with the proposed mechanism for 4-nitrophenyl acetate hydrolysis shown in Fig. 16 wherein the rate-determining step is attack on the substrate carbonyl. In this reaction, the best nucleophile will be free OH-. In metalloenzymes such as CA, the presence of the zinc center insures that a hydroxide nucleophile can be generated at relatively low concentrations of free OH-. 

In order to bypass the problem of designing a pocket from scratch, Bolon and Mayo [27] introduced a catalytically active His residue in thioredoxin, a well-defined 108-residue protein for which much structural and functional information was available. The design was based on the well-known reaction mechanism of p-nitrophenyl acetate hydrolysis and thioredoxin was redesigned by computation to accommodate a histidine with an acylated side chain to mimic transition state stabilization. The thioredoxin mutant was catalytically active and the reaction followed saturation kinetics with a k at of 4.6 x 10 s and a Km of 170 xM. The catalytic efficiency, after correction for differential protonation and nucleophilicity, can be estimated to be a factor of 50 greater than that of 4-methylimidazole, due to nucleophilic catalysis and proximity effects, see Section 5.2.3. 

If the a-chymotrypsin-catalysed hydrolysis of 4-nitrophenyl acetate [10] is monitored at 400 nm (to detect 4-nitrophenolate ion product) using relatively high concentrations of enzyme, the absorbance time trace is characterised by an initial burst (Fig. 5a). Obviously the initial burst cannot be instantaneous and if one uses a rapid-mixing stopped-flow spectrophotometer to study this reaction, the absorbance time trace appears as in Fig. 5b. Such observations have been reported for a number of enzymes (e.g. a-chymotrypsin [11], elastase [12], carboxypeptidase Y [13]) and interpreted in terms of an acyl-enzyme mechanism (Eqn. 7) in which the physical Michaelis complex, ES, reacts to give a covalent complex, ES (the acyl-enzyme) and one of the products (monitored here at 400 nm). This acyl-enzyme then breaks down to regenerate free enzyme and produce the other products. The dissociation constant of ES is k2 is the rate coefficient of acylation of the enzyme and A 3 is the deacylation rate coefficient. Detailed kinetic analysis of this system [11] has shown... 

In earlier work we had demonstrated that a zinc complex of pyridyl-2-carboxaldoxime (7) could be effective in cleaving esters. The interesting point is that the oxime anion is available as a nucleophile and the zinc as an electrophile, but they are not coordinated to each other - which would of course destroy the catalytic effect. To amplify catalysis we attached such oxime-zinc complexes to j8-cyclodextrin on both the secondary and primary faces of the cyclodextrin and examined their reaction with p-nitrophenyl acetate. We observed burst kinetics, in which there was an extremely rapid release of one mole of nitrophenoxide ion, followed by a slower release in a second phase. This indicated that we first rapidly produced the acetate of the oxime, and this then slowly hydrolysed to regenerate the oxime anion for further catalytic reaction. Such burst kinetics is very commonly seen in enzymatic reactions of para-nitrophenyl acetate, reflecting the same kind of two-step overall mechanism. 

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