Enzymatic Attack on Immobilized Substrates

1 l-Phenylalanine 4-nitroanilide was bound to agarose by applying the cyanogen bromide method. The chymotrypsin-catalyzed hydrolysis of the nitroanilide group was determined. As a spacer, one, two, three or four 6-aminohexanoyl residues were inserted between the gel matrix and the l-phenylalanine 4-nitroanilide. To compare the results obtained, the hydrolysis of soluble N-glutaryl-l-phenylalanine 4-nitroanilides was studied. 2 The hydrolysis of immobilized substrates is connected with chymotrypsin adsorption by the substrate gels. The enzyme adsorption could be described by the Langmuir isotherm. As characteristic parameters, constants for the affinity of the enzyme to the bound ligand (Ka) and for the maximum adsorption capacity of the substrate gel (Kmax) were determined. 3 In contrast to the soluble substrates, the enzymatic hydrolysis of the agarose-bound substrates was not complete. 4 The initial rates of the hydrolysis of the immobilized substrates was influenced by the spacer length. As the number of 6-aminohexanoyl residues in the spacer chain increased, so the agarosebound substrates were split with increasing velocity. 5 The initial rates of the hydrolysis of the substrate gels do not depend on the total enzyme concentration, but they are dependent on the amount of enzyme adsorbed by the gels. By correlating the reaction rate with the amount of adsorbed enzyme, the k′2 values could be determined as kinetic parameters. 6 An influence of diffusion on the course of the enzymatic hydrolysis of agarose-bound substrates could not be observed.