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 (K,) and for the maximum adsorption capacity of the substrate gel (Lax) 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 ki 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.Generally, heterogenously catalyzed enzyme reactions may be divided into two categories. The first comprises those in which the enzyme represents the solid phase, whereas the substrate is soluble. The second category includes the digestion of insoluble substrates by soluble enzymes. The reaction of an immobilized enzyme with a soluble substrate could be established as a biologically relevant model for corresponding enzyme reactions in vivo. Significant advances have been achieved in this field [1,2]. But only a little progress has been made in modelling enzymatic reactions with insoluble low-molecular-weight substrates [3].Abbreviations. PhMeSOs-, phenylmethanesulfony1-; Phe-Nan, L-phenylalanine 4-nitroanilide; Glt-Phe-Nan, N-glutaryl-L-phenylalanine 4-nitroanilide; Glt-(EAhx),Phe-Nan, N-glutary1-(6-aminohexanoyl),-L-phenylalanine 4-nitroanilide (n = 1, 2); (cAhx),-PheNan, (6-aminohexanoyl),-~-phenylalanine 4-nitroanilide (n = 1,2,3 and 4); A-= agarose-bound.Enzymes. Chymotrypsin (EC 3.4.21.1); trypsin (EC 3.4.21.4).In accordance with classic enzyme kinetics, the heterogenous reaction of a soluble enzyme with an insoluble substrate may be described byIn view of the great complexity of problems in such interface reactions, e.g. problems of diffusion, adsorption and difficulties in defining the concentration, the Michaelis-Menten equation cannot be used for soluble enzyme/insoluble substrate systems. Maclaren [4] has reported that the enzymatic hydrolysis of insoluble prot...
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