alpha-chymotrypsin and argininamide

The synthesis of the dipeptide N-benzoyl-l-tyrosine-l-argininamide (BTAA) was conducted under kinetic control with N-benzoyl-l-tyrosine ethyl ester as acyl donor and argininamide as nucleophile using immobilized α-chymotrypsin as catalyst. Using a mathematical procedure, the kinetic constants corresponding to the proposed mechanism of peptide synthesis were determined in three different cosolvent media, namely, ethanol, diglyme and acetonitrile. These constants were used for evaluating the selectivity of glyoxyl-agarose immobilized α-chymotrypsin in the synthesis of BTAA by determining the ratios of synthesis to hydrolysis rates.

Topics: Animals; Arginine; Biocatalysis; Cattle; Chymotrypsin; Dipeptides; Enzymes, Immobilized; Glyoxylates; Hydrolysis; Kinetics; Sepharose; Solvents; Tyrosine

We investigated the deacylation of two acyl-alpha-chymotrypsins by added nucleophiles. The nucleophile binding site of the enzyme shows a strong preference for positively charged compounds. Most of our data can be explained by direct electrostatic interaction between the ionic nucleophiles and two negatively charged residues which are located close to the active site of the enzyme molecule. The influence of inorganic salts on the acyl transfer includes the following effects: (1) reduction of electrostatic interactions between the acyl-enzyme and the nucleophile by addition of salts; (2) binding of divalent cations to the nucleophile binding site of the acyl-enzyme leading to a significantly changed specificity; and (3) linear dependence of the activity coefficients of the added nucleophiles on salt concentration.

Topics: Acylation; Arginine; Catalysis; Cations, Divalent; Cations, Monovalent; Chromatography, High Pressure Liquid; Chymotrypsin; Dipeptides; Electrochemistry; Electron Transport; Models, Chemical; Salts