The susceptibility of N-acetyl-S-alkyl- and N-acetyl-S-aryl-cysteine ethyl esters to chymotryptic hydrolysis.
ABSTRACT: 1. The preparation of the ethyl esters of some N-acetyl-S-alkyl- and N-acetyl-S-aryl-cysteine derivatives is described. 2. Kinetic parameters for the hydrolysis of these esters by chymotrypsin are reported. 3. The results are discussed in terms of the mechanism of action of chymotrypsin and also with reference to the possible application to sequence studies of cysteine-containing proteins.
Project description:1. The alpha-chymotrypsin-catalysed hydrolysis of N-acetylglycine ethyl and thiolethyl esters was investigated at pH7.90 and 25 degrees over a wide range of substrate concentrations. 2. The Lineweaver-Burk plots for these substrates are markedly curved, and it is shown that the curvature is due solely to the ;enzyme-blank' reaction. The rate of this reaction is proportional to free enzyme concentration in the range 10-100mum, with a pseudo-first-order rate constant of approx. 1x10(-3)sec.(-1). Correction for this reaction by the procedure described leads to linear plots. It is shown that the significance of the enzyme-blank reaction depends on the value of k(0)/K(m) for the substrate under investigation. 3. Interpretation of the curvature in the Lineweaver-Burk plots by previous workers in terms of activation by excess of substrate is shown to be erroneous. 4. Values of K(m) 387mm and k(0) 0.039sec.(-1), and K(m) 41mm and k(0) 0.23sec.(-1), were obtained for the ethyl and thiolethyl esters of N-acetylglycine respectively. The literature values for the methyl esters of N-acetyl- and N-propionyl-glycine have been corrected by the procedure described. The new values agree much better with current theories of alpha-chymotrypsin mechanism and specificity. 5. The kinetic parameters for the ethyl and thiolethyl esters indicate the absence of an electrophilic component in the catalytic mechanism of alpha-chymotrypsin, and the importance of the ester function in substrate binding.
Project description:Precise studies were performed on the effect of temperature on the rate and equilibrium parameters characterizing the individual stages of the alpha-chymotrypsin-catalysed hydrolysis of non-specific p-nitrophenol esters at pH 7.40 and 8.50. At both pH values the results indicate that a sharp kinetic anomaly is observed in Arrhenius plots of these parameters for the binding and acylation stages of the process, but not for the deacylation stage. Detailed comparison with other kinetic studies was made, and a comparison with thermal transitions observed in alpha-chymotrypsin by using physical techniques was attempted. A detailed discussion of possible causes of the anomalies is given.
Project description:1. A new method for synthesizing aryl esters of N-acylamino acids is described. The unsymmetrical anhydride resulting from the interaction of an N-acylamino acid and diphenylketen is allowed to react with a phenol. Cleavage of the anhydride by the phenol usually occurs in the desired direction. 2. Bacitracin has been examined as an enzyme model by determining its catalytic activity towards the hydrolysis of aryl esters. In general, it is less effective than imidazole. The variation of the catalytic constant with pH, together with other evidence, suggests that the histidine residue in bacitracin is the effective catalytic centre. 3. The stereoisomers of N-methoxycarbonylalanine p-nitrophenyl ester were hydrolysed at the same rate, but bacitracin was stereoselective towards the stereoisomers of the corresponding phenylalanine derivative.
Project description:A new transformation is presented that enables chemists to couple simple alkyl carboxylic acids with aryl zinc reagents under Ni-catalysis. The success of this reaction hinges on the unique use of redox-active esters that allow one to employ such derivatives as alkyl halides surrogates. The chemistry exhibits broad substrate scope and features a high degree of practicality. The simple procedure and extremely inexpensive nature of both the substrates and pre-catalyst (NiCl2·6H2O, ca. $9.5/mol) bode well for the immediate widespread adoption of this method.
Project description:A series of arylalkanoate esters and alpha-acetamidoarylalkanoate esters were tested as substrates for alpha-chymotrypsin and subtilisin BPN'. Chymotrypsin hydrolysed N-acetyl-l-phenylalanine methyl ester and methyl 4-phenylbutyrate faster than their respective higher and lower homologues, whereas methyl 2-acetamido-6-phenylhexanoate and methyl 6-phenylhexanoate were better substrates for subtilisin than their lower homologues. N-Acetyl-l-tryptophan methyl ester and its analogue, N-acetyl-3-(1-naphthyl)-alanine methyl ester, were hydrolysed 23 times faster by chymotrypsin than by subtilisin. These results indicate that the binding site of alpha-chymotrypsin is roughly 1.1nm (11A) long and curved, whereas that of subtilisin is a longer system and less curved. The stereo-specificity during the hydrolysis of typical substrates by both enzymes was found to vary over a wide range. The enhancing effect of the alpha-acetamido group in the l-series of substrates and the detrimental effect in the d-series of substrates also varies considerably.
Project description:A novel, metal-free aerobic oxidation method is described. 4-Dimethylaminopyridine (DMAP) successfully catalyzed the oxidation of aryl ?-halo esters to corresponding aryl ?-keto esters (up to 95% yield) under mild reaction conditions (Li2CO3, dimethylacetamide, air, and room temperature). A mechanism has been proposed where the oxidation proceeds through a [3 + 2] cycloaddition between O2 in an air atmosphere and pyridinium ylides. The ylides are supposedly generated from aryl ?-halo esters and DMAP in the presence of carbonates. Based on the plausible mechanism, the potential of DMAP as a catalyst in oxidation reactions was extended.
Project description:The partial purification and properties of an enzyme from the soluble fraction of rat liver that catalyses the reaction of glutathione with 2,3-unsaturated acyl thiol esters is described, and its possible role in the formation of S-carboxyalkylcysteines is discussed. The synthesis of S-(3-methylcrotonyl)- and S-(2-methylcrotonyl)-N-acetylcysteamine and of S-crotonyl-NN-dimethylcysteamine hydrochloride and dicyclohexylammonium S-crotonyl-N-acetyl-l-cysteine is described.
Project description:A novel synthetic methodology has been developed for the synthesis of diethyl 5-alkyl/aryl/heteroaryl substituted 3,4-dihydro-2H-pyrrole-4,4-dicarboxylates (also called 2-substituted pyrroline-4,5-dihydro-3,3-dicarboxylic acid diethyl esters) by iodide ion induced ring expansion of 2-[(aziridin-1-yl)-1-alkyl/aryl/heteroaryl-methylene]malonic acid diethyl esters in very good to excellent yields under mild reaction conditions. The electronic and steric impact of the substituents on the kinetics of ring expansion of N-vinyl aziridines to pyrrolines has been studied. Various diversely substituted novel pyrroline derivatives have been synthesized by this methodology and the products can be used as key intermediates in the synthesis of substituted pyrrolines, pyrroles and pyrrolidines.
Project description:Subtilisin BPN' hydrolysed N-acetyl-l-3-(2-naphthyl)-alanine methyl ester, N-acetyl-l-leucine methyl ester and N-acetyl-l-valine methyl ester, faster than alpha-chymotrypsin. Of eight ;locked' substrates tested, only methyl 5,6-benzindan-2-carboxylate was hydrolysed faster by subtilisin, whereas the other esters were better substrates for chymotrypsin. Compared with the values for chymotrypsin, the stereospecific ratios during the hydrolysis of the optically active locked substrates by subtilisin were decreased by one and two orders of magnitude for bi- and tri-cyclic substrates respectively. The polar groups adjacent to the alpha-carbon atom of locked substrates did not contribute significantly to the reactivity of the more active optical isomers, but had a detrimental effect on the less active antipodes during hydrolysis by both the enzymes. These studies show that the binding site of subtilisin BPN' is longer and broader than that of alpha-chymotrypsin.
Project description:The ?-trisphosphonic acid esters provide a unique spatial arrangement of three phosphonate groups and may represent an attractive motif for inhibitors of enzymes that utilize di- or triphosphate substrates. To advance studies of this unique functionality, a general route to alkyl derivatives of the parent system (R = H) has been developed. A set of new ?-alkyl-1,1,1-trisphosphonate esters has been prepared through phosphinylation and subsequent oxidation of tetraethyl alkylbisphosphonates, and the reactivity of these new compounds has been studied in representative reactions that afford additional examples of this functionality.