Synthesis and evaluation of a miniature organic model of chymotrypsin

D’Souza, Valerian T.; Hanabusa, Kenji; O’Leary, Timothy R.; Gadwood, Robert C.; Bender, Myron L.

doi:10.1016/0006-291x(85)91952-7

Cited by 60 publications

(14 citation statements)

References 10 publications

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“…3) (24,25). These compounds catalyze the hydrolysis of mtert-butylphenyl acetate at pH 10.7 with 15 being the better catalyst (26).…”

Section: B Cyclodextrin Imidazolesmentioning

confidence: 99%

“…Hilvert and Breslow investigatedˆrst 17, which did not catalyze benzoin condensation, but did catalyze deuteration of the aldehyde proton of benzaldehyde (28). Subsequently it was found that the g-cyclodextrin analogue 18 (22)(23) or ester hydrolysis (24)(25). In compound 25 the cyclodextrin is linked to a peptide, which structure is shown in single letter amino acid code.…”

Section: Cyclodextrins With Other Heterocyclic Groupsmentioning

confidence: 99%

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Cyclodextrin derivatives that display Enzyme Catalysis

Marinescu

Bols

2009

TIGG

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“…3) (24,25). These compounds catalyze the hydrolysis of mtert-butylphenyl acetate at pH 10.7 with 15 being the better catalyst (26).…”

Section: B Cyclodextrin Imidazolesmentioning

confidence: 99%

Section: Cyclodextrins With Other Heterocyclic Groupsmentioning

confidence: 99%

Cyclodextrin derivatives that display Enzyme Catalysis

Marinescu

Bols

2009

TIGG

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“…The synthesis of the artificial chymotrypsin (/3-benzyme) has been described elsewhere (5). The product was purified by reverse-phase preparative HPLC, and its purity was confirmed by HPLC, NMR, and thin-layer chromatography.…”

Section: Methodsmentioning

confidence: 99%

“…"'-Benzyme," an artificial chymotrypsin based on ,B-cyclodextrin, has been prepared. Its kinetics (kcat/Km) is equivalent to that of real chymotrypsin (5), and it has a turnover similar to that of a real enzyme (6).…”

mentioning

confidence: 99%

Thermal and pH stability of "beta-benzyme".

D’Souza¹,

Lu²,

Ginger³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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The thermal and pH stability of "I3-benzyme," an artificial chymotrypsin based on 13-cyclodextrin, has been studied and compared with the stability of real chymotrypsin. Artificial chymotrypsin is vastly superior to real chymotrypsin with regard to both temperature and pH stability. The reasons for this increased stability are discussed. Irreversible inactivation of chymotrypsin is brought about by disruption of the protein by cleavage of peptide linkages by the proton, by the hydroxide ion, or by the enzyme itself ("cannibalistic reaction") (3). It is thus important to study the stability of the enzyme and be able to overcome this deficiency. One of the important methods for increasing the stability of the enzyme is to immobilize the enzyme by covalently attaching it to a surface and thus prevent its intermolecular interactions and thereby minimize the cannibalistic reaction (4).Another method to avoid inactivation is to synthesize an artificial enzyme that is not susceptible to inactivation by the above-mentioned processes. "'-Benzyme," an artificial chymotrypsin based on ,B-cyclodextrin, has been prepared. Its kinetics (kcat/Km) is equivalent to that of real chymotrypsin (5), and it has a turnover similar to that of a real enzyme (6).With respect to its stability, it is important to note that artificial chymotrypsin contains no amino acids, whereas real chymotrypsin contains 245 amino acids. The function of 242 of the amino acids in the real enzyme is to achieve a precise and correct conformation through hydrogen bonding, while the other 3 amino acids are involved in catalysis. When one of the hydrogen bonds between these noncatalytic amino acids is disrupted, the conformation is changed and the enzyme activity diminishes. The disruption of the hydrogen bonds can be caused by extremes of temperature and pH, usually high temperature or high alkalinity. Any perturbation of the conformation that depends on amino acid interactions should be much less for the artificial enzyme than for the real enzyme. This has been observed in both thermal stability and pH stability (Figs. 1 and 2). EXPERIMENTALMaterials. The synthesis of the artificial chymotrypsin (/3-benzyme) has been described elsewhere (5). The product was purified by reverse-phase preparative HPLC, and its purity was confirmed by HPLC, NMR, and thin-layer chromatography. a-Chymotrypsin from Worthington was used without further purification. p-Nitrophenyl acetate was obtained from Aldrich and was recrystallized before use. m-(t-Butyl)phenyl acetate was synthesized from the corresponding phenol and acid chloride and recrystallized before use. All buffer solutions were prepared from doubly distilled water and analytical reagent grade chemicals. The pH values of all reaction mixtures and buffers were determined by pH meter (Altex model 3500 digital pH meter; Beckman).Kinetic Measurements. The kinetics of all the reactions were observed spectrophotometrically with a Cary 219 recording spectrophotometer (Varian) equipped with a thermostated cell compartme...

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“…In more elaborate approaches a 'substratebinding pocket' has been built or utilized, close to or around which the catalytic groups are arranged. Examples of the latter include the use of a solid matrix such as Sephadex [3], cyclodextrins [4,5] or 'spherands' [6].…”

Section: Introductionmentioning

confidence: 99%

The expression in E. coli of a polymeric gene coding for an esterase mimic catalyzing the hydrolysis of p‐nitrophenyl esters

Bülow

Mosbach

1987

FEBS Letters

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We have prepared a hybrid protein consisting of seven esterase units, Glu-Ala-His-Ala-Ser-Phe-Phe-Phe, fused to the N-terminal of galactokinase (E. cofi). The structural gene for this bifunctional protein was obtained by cloning a polymer made up of three chemically synthesized oligonucleotides to which the galactokinase gene was fused in frame. The hybrid protein was purified to homogeneity with the aid of the galactokinase moiety and showed an M, of 5 1000-53 000. The preparation could catalyze the hydrolysis of p-nitrophenyl esters and, due to the inbuilt hydrophobic spacers, Phe-Phe-Phe, improved catalysis of more hydrophobic substrates was obtained.

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Synthesis and evaluation of a miniature organic model of chymotrypsin

Cited by 60 publications

References 10 publications

Cyclodextrin derivatives that display Enzyme Catalysis

Cyclodextrin derivatives that display Enzyme Catalysis

Thermal and pH stability of "beta-benzyme".

The expression in E. coli of a polymeric gene coding for an esterase mimic catalyzing the hydrolysis of p‐nitrophenyl esters

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