Search

Several hydrophobic compounds acted as activators in dipeptide (Bz-Gly-L-Arg-OH, Z-Gly-L-Phe-OH) hydrolysis by bovine carboxypeptidase B. These hydrophobic compounds include Bz-Gly-OH, Z-Gly-OH, Z-L-Phe-OH, and Z-L-Phe-GLy-OH. These compounds were indicated to bind to the secondary substrate binding sites which is proposed to be responsible for substrate activation kinetics in dipeptide hydrolysis. Of the compounds Z-L-Phe-OH alone acted also as a inhibitor at higher concentrations, indicating that it binds to both primary and secondary sites as the dipeptide substrates do. Comparison of the activation effects of the compounds employed indicated that hydrophobic interaction played an important role in binding to the secondary site. Substrate and modifier binding constants were also determined and the results indicated that modifier binding increased both affinity and catalytic rate constant of the primary site. On the other hand, Z-Gly-OH and Z-L-Phe-Gly-OH inhibited the hydrolyses of tri and tetrapeptide substrates. This observation suggests that the secondary site is contained in the extended active center which the enzyme possibly has.

Catalytic and binding properties of bovine carboxypeptidase B were studied by kinetic and affinity chromatographic methods both using several oligopeptides as substrates or immobilized ligands. These oligopeptides contained either arginines or phenylalanines at carboxy termini as well as phenylalanyl residues in one of the other positions. The chromatographic studies showed that the phenylalanyl residues in endo-positions play a significant role in binding of the immobilized peptides to the enzyme, while the kinetics studies indicated further that the presence of an internal hydrophobic residue in a substrate was advantageous for the catalytic release of the carboxyl terminal residue from the substrate. These observations support the supposition that the enzyme has an extended active center which contains an extended hydrophobic binding site. Several hydrophobic compounds, which have been shown to act as activators in dipeptide substrate hydrolyses, showed inhibitory effect on hydrolyses of oligopeptide substrates. This observation suggests that these hydrophobic compounds bind to a portion of the hydrophobic site in the active center.

Affinity chromatography was used to characterize the binding properties of carboxypeptidase B with its ligands. The affinity adsorbents employed included arginine directly attached to agarose beads, arginine attached to the same support through hydrophilic and hydrophobic spacers, and immobilized caproylphenylalanine. The enzyme showed marked retardation on all of the arginine columns but only slight retardation on the phenylalanine column. Several hydrophobic ligands in solution enhanced to some extent the enzyme retardation on columns having arginine directly attached to the solid support, while several amino- and guanidino-alkylcarboxylic acids (lysine and arginine analogs) greatly enhanced the enzyme retardation on the phenylalanine column and somewhat enhanced it on the other columns having hydrophobic spacer arms. These observations confirmed that the enzyme has twobinding sites for the soluble and immobilized ligands and that these two sites exhibit cooperative ligations. Binding constants of the enzyme for various soluble ligands were also calculated from their chromatographic effects and the resulting values were interpreted in terms of the cooperative action of the two bindings sites, i.e., one for the primary binding of basic amino acid analogs (SITE I) and the other for hydrophobic compounds (Site II). In this chromatographic study, however, such cooperation of the two sites was obscure when arginine, acylarginine, or alkylarginine was the ligand directing to Site I.