alpha-chymotrypsin and 7-amino-4-methylcoumarin

Herein the reactivity of the enzyme α-chymotrypsin in the confinement of polystyrene-block-poly(acrylic acid) (PS-b-PAA) vesicles was investigated. Enzyme and substrate molecules were encapsulated in PS-b-PAA vesicles with internal diameters ranging from 26 nm to 165 nm during the formation of the vesicles. While the loading efficiencies of enzyme and substrate molecules were practically identical for vesicles of identical size, they were found to increase with decreasing vesicle size. The kinetics of the α-chymotrypsin catalyzed hydrolysis of N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) was evaluated following the increase of the absorption of the product 7-amino-4-methylcoumarin by UV/Vis spectroscopy. The values of the catalytic turnover number obtained for reactions inside vesicles with different sizes showed an increase of up to fourteen times compared to the bulk value with decreasing vesicle volume, while the values of the Michaelis-Menten constant decreased, respectively. This increase in reactivity of α-chymotrypsin is attributed to the effect of vesicle-wall interactions in the finite encapsulated space, where the reagents could diffuse, leading to enhanced collision frequencies.

Topics: Acrylic Resins; Biocatalysis; Chymotrypsin; Coumarins; Hydrolysis; Kinetics; Nanotechnology; Polymers; Polystyrenes; Spectrophotometry, Ultraviolet

This paper reports a novel and valuable approach to active-site titration. The starting substance for the preparation of the active-site titrants is aprotinin (bovine pancreatic trypsin inhibitor) in which the reactive-site peptide bond, Lys15-Ala16, is split. Two cystine disulfide bonds hold together the two peptide chains. The Lys15 of the reactive site is substituted by arginine-, phenylalanine- and valine-4-nitroanilide or by valine-7-amido-4-methylcoumarin. The different incorporated amino acid residues correspond to different specificities against serine proteinases. Serine proteinases with suitable specificity are able to remove 4-nitroaniline or 7-amino-4-methylcoumarin from these aprotinin derivatives while at the same time resynthesis of the reactive-site peptide bond occurs. The proteinase is then trapped in a stable enzyme-inhibitor complex, which prevents the proteinase from releasing further leaving groups. The quantity of 4-nitroaniline or 7-amino-4-methylcoumarin, which can be assayed spectrophotometrically or fluorometrically is equimolar to the quantity of proteinase used and trapped. The aprotinin derivatives with an incorporated Phe15 or Val15 residue are highly specific for chymotrypsin or for elastase from human leukocytes, respectively. The kinetic constants kon and koff of the enzyme-inhibitor complexes, and hence the equilibrium dissociation constants, can be calculated from the respective titration curves.

Topics: Alanine; Amino Acid Sequence; Amino Acids; Aniline Compounds; Aprotinin; Binding Sites; Chromatography, High Pressure Liquid; Chymotrypsin; Coumarins; Fluorometry; Humans; Indicators and Reagents; Kallikreins; Kinetics; Leukocytes; Lysine; Molecular Sequence Data; Pancreatic Elastase; Serine Endopeptidases; Spectrophotometry; Trypsin