alpha-chymotrypsin and benzoyltyrosine-4-nitroanilide

The reverse micelle (RM) media are very good as nanoreactors because they can create a unique microenvironment for carrying out a variety of chemical and biochemical reactions. The aim of the present work is to determine the influence of different water-dimethyl sulfoxide (DMSO) mixtures encapsulated in 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane RMs on the enzymatic hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide (Bz-Try-pNA) by α-chymotrypsin (α-CT). The reaction was first studied in homogeneous media at different DMSO-water mixture compositions and in DMSO-water/AOT/n-heptane RMs. The hydrolysis rates of Bz-Try-pNA catalyzed by α-CT were determined by UV-vis spectroscopy. The reaction follows the Michaelis-Menten mechanism and the kinetic parameters: kcat, KM, and kcat/KM were evaluated under different conditions. In this homogeneous media, DMSO plays an important role in the solubilization process of the peptide which is almost insoluble in water, but it has a tremendous impact on the inactivation of α-CT. It is shown that the enzyme dissolved in a 20% molar ratio of the DMSO-water mixture does not present enzymatic activity. Dynamic light scattering has been used to assess the formation of DMSO-water/AOT/heptane RMs at different DMSO compositions. The results also show that there is preferential solvation of the AOT RM interface by water molecules. To test the use of these RMs as nanoreactors, the kinetic parameters for the enzymatic reaction in these systems have been evaluated. The parameters were determined at fixed W(S) {W(S) = ([water] + [DMSO])/[AOT] = 20} at different DMSO-water compositions. The results show that the Michaelis-Menten mechanism is valid for α-CT in all the RM systems studied and that the reaction takes place at the RM interface. Surprisingly, it was observed that the enzyme encapsulated by the RMs show catalytic effects with similar kcat/KM values at any DMSO composition investigated, which evidence that DMSO molecules are localized far from the RM interface.

Topics: Animals; Cattle; Chymotrypsin; Dimethyl Sulfoxide; Heptanes; Hydrolysis; Kinetics; Micelles; Pancreas; Solutions; Succinates; Tyrosine; Water

Both activity and stability of alpha-Chymotrypsin (ChT) are substantially enhanced in the microdomains of laurylated or benzylated derivatives of poly(ethylenimine). EPR data revealed that the enhancement in activity of ChT is due to increase in the polarity of the microenvironment of Ser-195 caused by complexation of ChT to the polymer derivatives.

Topics: Benzyl Compounds; Chromatography, High Pressure Liquid; Chymotrypsin; Electron Spin Resonance Spectroscopy; Enzyme Activation; Hydrogen-Ion Concentration; Kinetics; Lauric Acids; Micelles; Models, Chemical; Polyethyleneimine; Tyrosine

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin, were investigated on the activation pathways of the human neutrophil respiratory burst. At 10 microM zLYCK, a parallel inhibition was observed of superoxide production stimulated with the chemo-attractant FMLP and of chymotrypsin-like activity of human neutrophils. By contrast, superoxide production induced by PMA was minimally affected by zLYCK. The known transduction pathways triggered by FMLP were analyzed. zLYCK did not affect either the FMLP-induced cytosolic free calcium transient, inositol 1,4,5 trisphosphate formation, nor the PMA-induced phosphorylation of the 47-kDa substrate of protein kinase C. zLYCK did not affect the activity of protein kinase C extracted from neutrophils. In Ca(2+)-depleted cells, in which phosphatidylinositol 4,5-biphosphate breakdown does not occur, zLYCK inhibited the FMLP-induced respiratory burst in cells primed by low doses of PMA. The activity of the NADPH oxidase tested with active membranes from stimulated neutrophils or in a cell-free system was not inhibited by zLYCK. We conclude that: 1) zLYCK inhibits superoxide production through the inhibition of a chymotrypsin-like protease of the neutrophil, 2) zLYCK inhibits FMLP-induced activation of NADPH oxidase through a pathway independent of PtdInsP2 breakdown and cytosolic free calcium, and 3) zLYCK may prove a useful probe for the characterization of its target protease in neutrophil activation.

Topics: Amino Acid Chloromethyl Ketones; Calcium; Chymotrypsin; Cytoplasm; Dose-Response Relationship, Drug; Enzyme Activation; Humans; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophils; Phagocytosis; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Superoxides; Time Factors; Tyrosine

In assaying chymotrypsin inhibition by the soybean Bowman-Birk inhibitor, two sequences of mixing the reactants were tried: adding the substrate last (s-last test) or adding the enzyme last (e-last test). The inhibition values obtained from the s-last test were either equal to or lower than those from the e-last test, depending on the pre-mix pH and pre-incubation time, while the values from the e-last test were independent of these conditions. The differences between the two tests were largest at a pre-mix pH of 4.0 or 8.5 and zero near pH 7.0. Abruptly changing the pre-mix pH from 4.0 to 7.0 brought the values of the two tests closer. These observations suggest a reversible, yet limited, hydrolysis of the inhibitor by the very enzyme it inhibits and indicate the greater reliability of the e-last test over the s-last test, paralleling a similar previous finding on the trypsin inhibition assay.

Topics: Chymotrypsin; Hydrogen-Ion Concentration; Indicators and Reagents; Trypsin Inhibitor, Bowman-Birk Soybean; Tyrosine

Potassium thiocyanate inhibited the activities of trypsin and chymotrypsin. The inhibition was mixed type on both enzymes with casein as substrate and on trypsin with tosyl-L-arginine methyl ester as substrate, but was uncompetitive on chymotrypsin with benzoyl-L-tyrosine p-nitroanilide as substrate.

Topics: Caseins; Chymotrypsin; Thiocyanates; Tosylarginine Methyl Ester; Trypsin; Trypsin Inhibitors; Tyrosine

Topics: Anilides; Blood; Chemical Phenomena; Chemistry; Chymotrypsin; Enzyme Inhibitors; Hydrolysis; Research; Tyrosine