alpha-chymotrypsin and glutarylphenylalanine-4-nitroanilide

The effect of human serum albumin (HSA) addition on the rate of hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) catalyzed by α-chymotrypsin has been measured in phosphate buffer saline at pH = 7.4. The presence of HSA (up to 200 μM) leads to a decrease in the rate of the process. The reaction follows a Michaelis-Menten mechanism under all the conditions employed. To take into account the effect of substrate depletion due to its binding to albumin ultrafiltration experiments were carried out from which the binding of GPNA to HSA was derived. After correction of the kinetic data taking into account the binding of GPNA to HSA, the activity of the enzyme, and the derived Michaelis constant and catalytic rate constant tends to remain almost independent of the presence of albumin, indicating that the depletion of the substrate due to its binding to HSA is the main factor affecting the enzyme activity.

Topics: Catalysis; Chymotrypsin; Dipeptides; Humans; Hydrolysis; Kinetics; Serum Albumin

The rate of N-glutaryl-L-phenylalanine p-nitroanilide hydrolysis catalyzed by alpha-chymotripsin has been measured in aqueous solutions of cetyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and dodecyltrimethylammonium bromide at concentrations below and above their critical micellar concentrations (CMC). For the three surfactants considered superactivity was observed, with maximum catalytic efficiencies taking place near the corresponding CMCs. The effect of the surfactants after the CMCs is mostly due to a decreased thermodynamic activity of the substrate due to its incorporation into the micelles. After addition of the surfactants, the Michaelis constant values (corrected to take into account the free substrate concentration) tend to decrease, passing through an ill defined minimum, afterwards reaching a constant value. The catalytic rate constants show the same profiles that the catalytic efficiency, being maxima near the surfactants CMCs. This maximum is more important for the surfactant having the shorter tail. This result is explained by considering that the hydrophobicity of the surfactant influences more the CMC than its association to the enzyme.

Topics: Catalysis; Chymotrypsin; Dipeptides; Hydrolysis; Kinetics; Quaternary Ammonium Compounds

The rate of hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) catalyzed by alpha-chymotrypsin (alpha-CT) has been measured in aqueous solutions of dodecyltrimethylammonium bromide (DTAB) at concentrations below and above the critical micelle concentration, as well as in the absence of surfactant. Under all the conditions employed, the reaction follows a Michaelis-Menten mechanism. The presence of the surfactant leads to superactivity below and above the critical micelle concentration (CMC), with a maximum reaction rate taking place near the CMC when the results are treated in terms of the analytical concentration of the substrate. A similar behavior was observed by working with the enzyme partially deactivated in the presence of 4 M urea. After correction to take into account the partitioning of the substrate between the micelles and the external media, the activity of the enzyme tends to remain almost constant above the corresponding CMCs. This results from a compensation of a decrease in the catalytic constant (k(cat)) and a decrease in the Michaelis constant (K(M)). The behavior of alpha-CT in the hydrolysis of GPNA in DTAB solutions is at variance with that previously reported for the hydrolysis of 2-naphthyl acetate in solutions of the same surfactant (E. Abuin, E. Lissi, R. Duarte, Langmuir 19 (2003) 5374). An explanation of the different effects of the surfactant on the behavior of the enzyme with both substrates is advanced, taking into account the complexity of the mechanism of the alpha-CT-mediated reaction, more specifically, in terms of different rate-limiting steps for the formation of the measured products.

Topics: Catalysis; Chymotrypsin; Dipeptides; Hydrolysis; Kinetics; Quaternary Ammonium Compounds; Solutions; Water

A novel and facile method for the preparation of an enzyme-immobilized microreactor has been developed in which enzymes are immobilized as an enzyme-polymer membrane formed on the inner wall of the microchannel by a cross-linking polymerization method; the resulting microreactor shows excellent reaction performance and stability against denaturating agents.

Topics: Biosensing Techniques; Chymotrypsin; Cross-Linking Reagents; Dipeptides; Enzymes, Immobilized; Hydrolysis; Polytetrafluoroethylene; Substrate Specificity

Topics: Adult; Animals; Bacteria; Benzoylarginine Nitroanilide; Case-Control Studies; Chymotrypsin; Culture Media; Dental Plaque; Dipeptides; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Endopeptidases; Female; Humans; Male; Middle Aged; Milk; Oligopeptides; Pancreatic Elastase; Periodontal Index; Periodontitis; Porphyromonas gingivalis; Prevotella intermedia; Substrate Specificity; Trypsin

The Michaelis constant of alpha-chymotrypsin, immobilized on a glutaraldehyde-activated silicate support, for N-glutaryl-L-phenylalanine-p-nitroanilide was determined and was found to be identical with that of the enzyme in solution. The influence of intraparticular diffusion was taken into account by immobilizing different amounts of enzyme, thus changing the magnitude of diffusional constraints and extrapolating apparent Michaelis constants, determined for each amount of immobilized enzyme, to zero diffusional constraints. The possible effect of the immobilized enzyme distribution inside the porous matrix was investigated through numerical simulations.

Topics: Adsorption; Chymotrypsin; Diffusion; Dipeptides; Enzymes, Immobilized; Glutaral; Kinetics; Silicic Acid

Topics: Aldehydes; Animals; Caseins; Cattle; Chemical Phenomena; Chemistry; Chymotrypsin; Chymotrypsinogen; Dipeptides; Isoelectric Focusing; Kinetics; Structure-Activity Relationship; Thermodynamics; Tyrosine