Artificial chymotrypsin

A similar concept was used in the development of artificial chymotrypsin mimics [54]. The esterase-site was modeled by using the phosphonate template 75 as a stable transition state analogue (Scheme 13.19). The catalytic triad of the active site of chymotrypsin - that is, serine, histidine and aspartic acid (carboxy-late anion) - was mimicked by imidazole, phenolic hydroxy and carboxyl groups, respectively. The catalytically active MIP catalyst 76 was prepared using free radical polymerization, in the presence of the phosphonate template 75, methacrylic acid, ethylene glycol dimethacrylate and AIBN. The template removal conditions had a decisive influence on the efficiency of the polymer-mediated catalysis, and best results were obtained with aqueous Na2CC>3. 

The catalytic activity of artificial chymotrypsin in the hydrolysis of m-tert-butylphenyl acetate (k = 2.8xl02 s 1, KM = 13xl05M) was found to be close to the activity of chymotrypsin in the hydrolysis of p-nitrophenyl acetate (k,.at = l.lxlO2 s 1, KM = 4x105M). Another example of mimicking enzyme catalysis by P-cyclodextrin is general acid-base-catalyzed hydrolysis and nitrosation of amines by alkyl nitrites (Iglesias, 1998). 

Figure 6..9. The mechanism of action of artificial chymotrypsin (a) Complete model of acylchymotripsin (a) and miniature organic model of chymotrypsin (b) (D Souza and Bender, 1987). Reproduced with permission.

ABSTRACT. The synthesis and characterization of an artificial acyl-enzyme intermediate of chjrmotrypsin, and an artificial enzyme, chymotryp-sin, are described. They both contain the same three catalytic groups, an imidazolyl group, a hydroxyl group, and a carboxylate ion. In the acyl-enzyme, the three groups are attached to a norbornane backbone, but in the artificial enzyme, the three catalytic groups are attached to a cycloamylose as binder. The artificial acyl-enzyme shows a rate of hydrolysis 154,000 times faster than an ordinary ester and only 18-fold slower than the real acyl-chymotrypsin. The artificial chymotrypsin is over a thousand fold slower than real chymotrypsin, presumably because of impurities in the preparation. 

Figure 6. Theoretical synthesis of artificial chymotrypsin. practically in Figure 7. That is to say, if the catalytic groups are...

Figure 8. Artificial chymotrypsin-catalyzed hydrolysis of -t-butyl-phenyl trimethylacetate at 25° in acetonitrile-water C50% v/v) at pH 10.5 borate buffer [E]q = 1x10 M [S]q = 1x10 M.

The two kinetic steps seen in artificial chymotrypsin catalysis imply that the mechanism of action of the artificial enzyme is as shown in Figure 9 (for the corresponding acetate ester). One obvious experiment yet to do is to prove that the acetyl group becomes attached to the cycloamylose by use of a radioactive acetate ester. 

The impure, artificial chymotrypsin we have thus far produced is only about 1/1000 as good as real chymotrypsin as Table III will show. 

Thus, the impurity does not allow us to claim that we have made an artificial chymotrypsin, alt cjugh we can claim that we have made artifi-... 

Much of what is known about the chymotrypsin mechanism is based on studies of the hydrolysis of artificial substrates—simple organic esters, such as /Miitrophenylacetate, and methyl esters of amino acid analogs, such as... 

E.V. Kudryashova, A.K. Gladilin, A.V. Vakurov, F. Heitz, A.V. Levashov, and V.V. Mozhaev, Enzyme-poly electrolyte complexes in water-ethanol mixtures negatively charged groups artificially introduced into alpha-chymotrypsin provide additional activation and stabilization effects. Biotechnol. Bioeng. 55, 267-277 (1997). 

ACE inhibitory peptides have been separated from the skin of skate by Lee et ah (2011). The purified peptides showed IC50 values of 95 and 148 M, respectively, for both peptides isolated from the skin of skate. Further, Lineweaver-Burk plots indicated that the peptides act as noncompetitive inhibitor against ACE. Recently, many inhibitory peptides against ACE are reported (Table 15.2) as natural alternative biofunctional peptides that are safer than that of the existing artificial ACE inhibitory compounds in the market that show some side effects. Various peptides have been isolated from seafood by-products from the fisheries industry such as backbones from tuna (Lee et al., 2010). Tuna backbone has hydrolyzed using various proteases such as alcalase, a-chymotrypsin, neutrase, papain, pepsin, and trypsin to obtain an antioxidant peptide (Je et ah, 2007). 

We have made several artificial enzymes that use cyclodextrin to bind a substrate and then react with it by acylating a cyclodextrin hydroxyl group. This builds on earlier work by Myron Bender, who first studied such acylations [83]. We added groups to the cyclodextrin that produced a flexible floor, capping the ring [84]. The result was to increase the relative rate of cyclodextrin acylation by m-t-butylphenyl acetate from 365 relative to its hydrolysis rate in the buffer to a Complex/ buffer of 3300. We changed the substrate to achieve better geometry for the intracomplex acylation reaction, and with a p-nitrophenyl ester of ferroceneacrylic acid 10 we achieved a relative rate for intracomplex acylation of ordinary [3-cyclodextrin vs. hydrolysis of over 50 000 and a Vmax comparable to that for hydrolysis of p-nitrophenyl acetate by chymotrypsin... 

I Cyclodextrins are excellent enzyme models Catalysis and induced fit. Due to their cavities, which are able to accommodate guest (substrate) molecules, and due to the many hydroxyl groups lining this cavity, cyclodextrins can act catalytically in a variety of chemical reactions and they therefore serve as good model enzymes. Thus, benzoic acid esters are hydrolyzed in I aqueous solution by factors up to 100 times faster if cyclodextrins are added. The reaction in- j volves an acylated cyclodextrin as intermediate which is hydrolyzed in a second step of the j reaction, a mechanism reminiscent of the enzyme chymotrypsin. The catalytic efficiency can. be further enhanced if the cyclodextrins are suitably modified chemically so that a whole range of artificial enzymes have been synthesized [551-555, 556, 563, 564]. 

Phage libraries have also been used to study the substrate specificity of enzymes by finding an improved artificial substrate. Coombs et al. (69) reported the detailed assessment of specificity for a serine protease belonging to the a-chymotrypsin family, the prostate specific antigen (PSA). They used both substrate optimization by singlepoint mutations and phage display libraries. The sequence of the 14-member substrate 10.2 (70) was used to start the iterative optimization process (Fig. 10.11) in which substitution or exchange of the PI, P2, or P2 residues increased the substrate affinity... 

Acylation of fl-cyclodextrin. The acylation of /5-cyclodextrin is modestly accelerated by bound m-nitrophenyl acetate, m-N02C6H40C0CH3. Recently acylation of /3-cyclodextrin at a rate comparable to acylation of chymotrypsin has been reported. The acylating reagent is the p-nitrophenyl ester (1) of ferrocinnamic acid. This reagent was chosen because ferrocene is strongly bound within the cavity of /3-cyclodextrin. The acylation is accelerated by > 50,000-fold compared to hydrolysis of 1 in DMSO-HjO alone buffered at pH 6.8. Thus cyclodextrins can behave as artificial enzymes. 

Aspartame monitoring represents a second interesting task for the ET. Immobilized a-chymotrypsin hydrolyzes the artificial sweetener under release of protons which are easily detected via a tris/HCl-buffer due to its high protonation enthalpy. The immobilized enzyme unfortunately causes an unspecific conversion. However, the procedure might be interesting for monitoring aspartame during its industrial production within the Tosoh-process. 

A much more improved antibody catalyst for amide hydrolysis has been elicited very recently by a joint hybridoma and combinatorial antibody library approach. The measured with a primary amide substrate at pH 9 and 25 °C was 5x10" s" for this new antibody. This corresponds to a half-life of 4 h when the substrate is fully complexed to the active site. The half-lives for the amide hydrolysis catalyzed by the antibodies are much longer than that (10-30 min at pH 4.5-7 and 4 °C when the substrate is fully complexed to the active site) of the light chain of Gbn hydrolyzed by 39. The fastest protein cleavage recorded so far with artiHcial proteinases is the cleavage of chymotrypsin by a coordinatively polymerized bilayer membrane which will be discussed later in this review. The coordinatively polymerized bilayer membrane achieved half-life as short as 3 min at 4 °C and pH 5.5-9.5. This is several times faster than the hydrolysis of Gbn by 39. In terms of utility in practical applications, however, 39 is more useful than the artificial enzyme based on the bilayer membrane due to the immobile nature of the former as well as the intrinsic instability of bilayer membranes. 

C. A. Venanzi and J, D. Bunce, hit. j. Quantum Chem. Quantum Biol. Symp., 12, 69 (1986). Molecular Recognition by Artificial Enzymes Cyclic Urea Minietics of Alpha Chymotrypsin. 

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