Chymotrypsin enzymatic hydrolysis

Partial hydrolysis of a peptide can be carried out either chemically with aqueous acid or enzymatically. Acidic hydrolysis is unselective and leads to a more or less random mixture of small fragments, but enzymatic hydrolysis is quite specific. The enzyme trypsin, for instance, catalyzes hydrolysis of peptides only at the carboxyl side of the basic amino acids arginine and lysine chymotrypsin cleaves only at the carboxyl side of the aryl-substituted amino acids phenylalanine, tyrosine, and tryptophan. 

One of the earliest suggestions that total enzymatic hydrolysis was possible came from the studies of Frankel (1916), who showed that over 90 % of the bonds in several proteins could be broken when proteolysis with pepsin, trypsin, and chymotrypsin was followed by prolonged hydrolysis with the erepsin preparation of Cohnheim (1901). The recognition in later years of several peptidases in intestinal exti acts which will specifically act upon bonds that are not susceptible to the endopoptidases (Bcrg-mann, 1942) probably accounts for these obseiwations. The specific peptidases such as prolidase, iminodipeptidase (prolinase), glycylglycine dipeptidase, tripeptidase, and leucine aminopeptidase, whi( h are present in mucosa, attack many of the bonds that resist the action of endopoptidases. 

The known desymmetrization of prochiral 3-substituted glutarates via enzymatic hydrolysis [65] has been optimized by chemists at Ciba Speciality Chemicals for the synthesis on a large scale [66]. The a-chymotrypsin-catalyzed process is characterized by a high substrate concentration of 285 g L and an isolated yield of 94% product with an ee of 98.2% (route C). 

Furthermore, enzymatic hydrolysis of model isopeptides N -oligo(L-methionyl)-l-lysine from Bio-beads13031 by pepsin, chymotrypsin, cathepsin C (dipeptidyl peptidase IV) and intestinal aminopeptidase N was investigated using high-performance liquid chromatography to identify and quantify the hydrolysis products 3041. 

Considerable attention has also been given to enantioselective enzymatic hydrolysis of esters of a-amino acids. This is of particular importance as a means of preparing enantiopure samples of unusual (non-proteinaceous) a-amino acids. The readily available proteases a-chymotrypsin (from bovine pancreas) and subtilisin (from Bacillus lichenformis) selectively hydrolyze the L-esters, leaving D-esters unreacted. These enzymatic hydrolysis reactions can be applied to V-protected amino acid esters, such as those containing r-Boc and Cbz protecting groups. 

We have shown by a comparison of the pH dependence of the step characterized by ki that the hydrolysis of the enzyme-acyl compound is the rate-determining step for the enzymatic hydrolysis of the usual amino acid amide substrates. In the case of chymotrypsin, acetyl-L-phenylalanine ethyl ester is hydrolyzed 1,000 times faster than the corresponding amide and in the case of trypsin, benzoyl-L-arginine ethyl ester is hydrolyzed 300 times faster than the corresponding amide. This suggests that for the amide hydrolysis too the second step, the acylation of the enzyme, must be the rate-determining step, since the third step is obviously identical for esters and amides of the same amino acid derivatives. The pH dependence of the chymotrypsin-catalyzed hydrolysis of acetyl-L-tyrosine ethyl ester and acetyl-L-phenylalanine ethyl ester indicates that for these reactions ki and kz are of the same order of magnitude and both contribute to the over-all rate, as shown by Equation (4). 

Kopecek J, Rejmanova P, Chytry V. Polymers containing enzymatically degradable bonds. 1. Chymotrypsin catalyzed hydrolysis of p-nitroanilides of phenylalanine and tyrosine attached to sidechains of copolymers of N-(2-hydroxypropyl)-methacrylamide. Makromol Chem 1981 182 799-809. 

The pH dependence of enzymatic activity in microemulsions sometimes differs from that in aqueous solution. For instance, several groups found that for a-chymotrypsin-catalyzed hydrolysis the optimum pH is shifted to considerably more alkaline conditions in the... 

Enzyme reaction intermediates can be characterized, in sub-second timescale, using the so-called pulsed flow method [35]. It employs a direct on-line interface between a rapid-mixing device and a ESI-MS system. It circumvents chemical quenching. By way of this strategy, it was possible to detect the intermediate of a reaction catalyzed by 5-enolpyruvoyl-shikimate-3-phosphate synthase [35]. The time-resolved ESI-MS method was also implemented in measurements of pre-steady-state kinetics of an enzymatic reaction involving Bacillus circulans xylanase [36]. The pre-steady-state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase were determined. The MS results were in agreement with those obtained by stopped-flow ultraviolet-visible spectroscopy. In a later work, hydrolysis of p-nitrophenyl acetate by chymotrypsin was used as a model system [27]. The chymotrypsin-catalyzed hydrolysis follows the mechanism [27] ... 

ABSTRACT. Cyclodextrin (CD) has a hydrophobic cavity which acts like a binding site of an actual enzyme. But enzymatic turnover reaction did not occur in CD-catalyzed reactions.3-CD was modified by a histamine group to attach a reactive functional group. 3-CD-histamine accelerates the hydrolysis of p-nitrophenyl acetate. Catalytic rate constant of this reaction is close to an actual enzyme, a-chymotrypsin. Enzymatic turnover reaction is realized with this compound at around neutral pH value. 

One of the most desired transformations is a hydrolysis of above substances resulting in free lysergic acid, the substrate for many semisynthetic preparations. Chemical hydrolysis of its derivatives gives low yields (50-65%). Fermentative production of lysergic acid is somehow complicated. Enzymatic hydrolysis of peptide alkaloids is still impracticable. Common proteolytic enzymes (papain, subtilisin, chymotrypsin, termolysin) do not attack peptidic bond in ergokryptine... 

This method involves equimolar amounts of (I) and (II), and organic base as the acceptor of liberated p-toluenesulfonic acid. Polymers with high molecular weights, and yields froiji 68 to 95% (depending on mol. weights) were characterized by GPC, H NMR spectroscopy and DSC. Their enzymatic hydrolysis (lipase, a- chymotrypsin) was studied. 

When a fresh solution of the R,S conformer (in 95 5 water-dioxane) is incubated with a-chymotrypsin no hydrolysis occurs. However, with an aged stock solution in dioxane a-chymotrypsin-catalyzed hydrolysis takes place readily. Hence, isomerization to an enzymatically active conformer is gradually taking place upon destruction by dissolution in an organic solvent of the crystal structure of the R,S conformer. 

Also important is the finding that not only the conformer is inert to hydrolysis by a-chymotrypsin, but it also failed to inhibit enzymatic hydrolysis of the active S,Seq conformer. In marked contrast, l-KCTI has been shown to strongly inhibit chymotryptic hydrolysis of d-KCTI. This pattern of competitive inhibition has also been demonstrated for other enantiomeric pairs of chymotrypsin substrates. To understand this behavior it should be realized that the two conformers of Belleau s compound differ in two important aspects orientation of the carbomethoxyl group and the chirality of the biphenyl system. Consequently, it must be concluded that in its reaction with this constrained substrate, a-chymotrypsin displays specific recognition of molecular asymmetry. This is referred to as tertiary structural specificity. The specificity of the biphenyl compound thus serves to extend the concept that appropriately constrained substrates can serve as very useful tools. 

The enzymatic hydration of lactones is also documented, a variety of hydrolases having demonstrated activity. Very detailed kinetic studies have, for example, been published on the hydrolysis of oxazolones (7.78, R = H or Me, R = Me or aryl, R" = Me or Ph) catalyzed by a-chymotrypsin [163], These compounds are interesting from a chemical point of view, being enolic lac-... 

Biocatalysis plays a central role in the manufacturing of statin side chains (Figure 6.2). A first set of approaches exploits enzymatic desymmetrization reactions, for example, of the methoxyacetyl ester of glutaric acid diethyl ester with commercially available a-chymotrypsin as explored by Ciba SC with a yield of 94% and enantiomeric excess of up to 98% [1]. In the optimized procedure, the substrate was available in a concentration of 1 M at an enzyme/substrate ratio of 7% (wt/wt), and the reaction took approximately a day. The subsequent steps to the final acetonide also involved a pig-liver esterase (PLE) catalyzed selective hydrolysis of the methoxyacetyl group (Figure 6.2a). 

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