Trypsin ester hydrolysis

Trypsin Ester Hydrolysis CTAB/chlorof m/isooctane [74]... 

K, Tanizawa, Y Kasaba, and Y. Kanaoka- Inverse substrates" for trypsin. Efficient hydrolysis of certain esters with a cationic center in the leaving group J. Am. Chem. 

Fig. 3. Time course of the trypsin-catalyzed hydrolysis of p-amidinophenyl acetate (46 R = CH3) at pH 8.0, 25 °C. Concentrations of enzyme and ester are 10 pM and 0.7 mM, respectively...

Considerable effort has been applied to studies of ester hydrolysis catalyzed by imidazoles (76MI40700, 80AHC(27)241). Certainly, 1-acetylimidazole can be made enzymically, probably by the sequence acetyl phosphate + coenzyme A acetylcoenzyme A+phosphate, acetyl-coenzyme A + imidazole l-acetylimidazole+coenzyme A. In addition, the imidazolyl group of histidine appears to be implicated in the mode of action of such hydrolytic enzymes as trypsin and chymotrypsin, thereby engendering further interest in the process of imidazole catalysis. The two pathways which have been found to be involved are general base catalysis and nucleophilic catalysis. In the former (Scheme 26) a basic imidazole molecule can activate a water molecule to attack the ester at the carbonyl carbon, this being followed by the usual sequence of steps as in simple hydroxide ion hydrolysis. At high imidazole concentrations the imidazole molecules may be involved directly. 

For example, a superb mimic of the charge-relay system in serine proteases has been prepared by attaching both carboxylate and imidazole to a-, fi-, and y-CyDs [24]. The hydroxy group, the last component of the charge-relay system, is provided by the CyDs. The activity (kinetic parameters) of the -CyD-based artificial enzyme for ester hydrolysis is close to that of aartificial enzymes show acylation, deacylation, and turnover, as is observed in the reactions of chymo-trypsin. The substrate-specificity is dependent on the kind of CyD used, since it is primarily governed by the substrate-binding process. In phenyl ester hydrolysis, a- and yS-CyD-based artificial enzymes are better than the y-CyD-based artificial enzyme. For the hydrolysis of tryptophan ethyl ester, however, the y-CyD-based artificial enzyme is the best. In another serine protease model, tripeptide (Ser-His-Asp) is directly introduced to the primary hydroxyl side of f -CyD [25]. This... 

The trypsin catalyzed hydrolysis (Fig. 7A) of the highly specific substrate Na carbobenzyloxy-L-lysine-ji-nitrophenyl-ester (Z-lys-pNP) has been studied in detail under cryoenzymological conditions by both spectrophotometry and NMR spectroscopy. The kinetic data from both techniques confirm that the kinetics and mechanism under cryoenzymological conditions are essentially the same as those determined at ambient temperatures by rapid reaction techniques. The hydrolysis of [ C]Z-lys-pNP[(S) in Fig. 7A, 8 = 173.6 ppm] by trypsin was monitored by the decrease in intensity of this signal and the increase in the signal arising from the product (P2 in Fig. 7A, 8 = 177.7... 

Trypsin Protein hydrolysis (for details see industrial section above) Inflammation Spectrophotometry or microcalorimetry One BAEE (Af-benzoyl-L-arginine ethyl ester) unit is an absorbance change at 253 nm of 0.001 per min with BAEE as substrate at pH 7.6 and 25°C... 

Horseradish peroxidase Trypsin (protease) Subtilisin (protease) Phenol polymerization Transpeptidation Ester hydrolysis Ethylacetate Butan-l,4-diol Dioxane, chloroform, etc. 

In the field of organic synthesis, it was reported that the catalytic hydrolysis of umbelliferone esters (7-acetoxycoumarin) to 7-hydroxycoumarinby porcine pancreatic lipase covalently immobilized on microchannel reactors almost completed within 1 min, to be compared with 4 min in a normal batch reaction [86]. The same group demonstrated an improvement in the yield of trypsinmicrochannel reactor with a lower enzyme concentration but a 20-fold higher reaction rate than in a batch reactor [87]. 

Trypsin Ester, amide hydrolysis AOT/isooctane and CTAB/ chlaroform/isooct e [71]... 

The parameters are usually obtained from a series of initial rate experiments performed at various substrate concentrations. Data for the hydrolysis of benzoyl-L-tyrosine ethyl ester (BTEE) by trypsin at 30 V and pH 7.5 are given below ... 

Procarboxypeptidase A is activated by the removal of a peptide of some 64 residues from the N-terminus by trypsin.153 This zymogen has significant catalytic activity. As well as catalyzing the hydrolysis of small esters and peptides, procarboxypeptidase removes the C-terminal leucine from lysozyme only seven times more slowly than does carboxypeptidase. Also, the zymogen hydrolyzes Bz-Gly-L-Phe with kcsA = 3 s-1 and KM = 2.7 mM, compared with values of 120 s 1 and 1.9 mM for the reaction of the enzyme.154 In contrast to the situation in chymotrypsinogen, the binding site clearly pre-exists in procarboxypeptidase, and the catalytic apparatus must be nearly complete. 

Catalytic tests were performed in a glass vessel equipped with a stirrer motor. Two monoliths (diameter 4.3 cm, length 4 cm) were mounted in plane on the stirrer axis. The total reaction volume was 2.5 1. Lipase was assayed in the acylation of vinyl acetate with butanol in toluene. Initial reaction rate was followed by GC analysis. Immobilized trypsin was used in the hydrolysis of N-benzoyl-l-arginine ethyl ester (BAEE) in a 0.01 M phosphate buffer pH 8 at 308 K. The reaction was followed by UV-VIS at 253 nm, and reaction rate was calculated in the mass transfer limited situation. 

The structural analysis of the trypsin inhibitor from bovine pancreas (BPTI) in complex with trypsin shows that the inhibitor occupies and blocks the substrate binding pocket in a highly complementary maimer (fig. 2.9). In the trypsin-BPTI complex, the catalytically essential Ser-OH of trypsin contacts a CO group of the inhibitor in a manner very similar to the tetrahedral transition state of amide or ester bond hydrolysis (see fig. 2.9b). The inhibitor can be likened to a pseudo-substrate and, as such, is bound with high affinity. The cleavage of the peptide bond is, however, not possible due to other circumstances, such as the fact that water is prevented from reaching the active site with the inhibitor boimd. 

Various esterases exist in mammalian tissues, hydrolyzing different types of esters. They have been classified as type A, B, or C on the basis of activity toward phosphate triesters. A-esterases, which include arylesterases, are not inhibited by phosphotriesters and will metabolize them by hydrolysis. Paraoxonase is a type A esterase (an organophosphatase). B-esterases are inhibited by paraoxon and have a serine group in the active site (see chap. 7). Within this group are carboxylesterases, cholinesterases, and arylamidases. C-esterases are also not inhibited by paraoxon, and the preferred substrates are acetyl esters, hence these are acetylesterases. Carboxythioesters are also hydrolyzed by esterases. Other enzymes such as trypsin and chymotrypsin may also hydrolyze certain carboxyl esters. 

Carboxybiotin. The structure of biotin suggested that bicarbonate might be incorporated reversibly into its position 2. However, this proved not to be true and it remained for F. Lynen and associates to obtain a clue from a "model reaction." They showed that purified P-methylcrotonyl-CoA carboxylase promoted the carboxylation of free biotin with bicarbonate (H14C03 ) and ATP. While the carboxylated biotin was labile, treatment with diazomethane (Eq. 14-6) gave a stable dimethyl ester of N-l -carboxybiotin.53 54 The covalently bound biotin at active sites of enzymes was also successfully labeled with 14C02 Treatment of the labeled enzymes with diazomethane followed by hydrolysis with trypsin and pepsin gave authentic N-l -carboxybiocytin. It was now clear that the cleavage of ATP is required to couple the C02 from HCOs to the biotin to form carboxybiotin. The enzyme must... 

Preparation of Human Insulin. Porcine insulin can be converted to the human insulin sequence by an enzyme-catalyzed transpeptidation reaction (10,11). Under appropriate conditions trypsin acts preferentially at LysB29 rather than ArgB22 to yield a covalent des[B30]insulin/trypsin complex (acyl—enzyme intermediate). In the presence of high concentrations of organic co-solvents and the /-butyl ester of threonine, transpeptidation predominates over hydrolysis to yield the /-butyl ester of human insulin. Following appropriate purification steps and acidolytic removal of the ester, human insulin suitable for treating patients is obtained. 

In Table II are shown the results from kinetic studies with commercially available gastric and pancreatic enzymes. Trypsin was strongly inhibited, at least at a low concentration of casein as substrate. The hydrolysis of benzoyl arginine ethyl ester (BAEE) by trypsin was non-competitively inhibited, giving a 30% reduction of Vmax at 0.5 mg/ml of the LMW fraction. Carboxypepti-dase A, and to a lesser extent carboxypeptidase B, were non-competitively inhibited as well. Pepsin and chymotrypsin were not affected by the conditions used in these assays. 

The substrate specificity of many esterases is not high (19) and the same is true of some proteases (amide-hydrolyzing enzymes), such as a-chymo-trypsin (12, 20). Amides may also serve as substrates for some esterases (21). Since esterases and proteases are widespread, hydrolysis of ester or amide linkages often accompanies other transformations by intact organisms. Soluble hydrolases are often present in supernatant fractions of mammalian microsomal preparations, and hydrolytic reactions may also occur when extracts of this type are used. Glycosidases, which catalyze the hydrolysis of... 

---