The kinetic parameters Km and kcat/Km have been determined for the carboxypeptidase B (CPB, EC 3.4.12.3) catalyzed hydrolysis of benzoylglycyl-DL-homolysine and benzoylglycyl-L-homoarginine. Plots of these data and those for Bz-Gly-Orn and Bz-Gly-Arg (Wolff, E. C., Schirmer, E. W. &Folk, J. E. (1962) J. Biol. Chem. 237, 3094–3099) and Bz-Gly-Lys versus the length of the side chain of the basic amino acid indicate that unlike trypsin (EC 3.4.21.4) (Seely, J. H. &Benoiton, N. L. (1970) Can. J. Biochem. 48, 1122–1131) CPB has a higher binding affinity for a guanidino group than for an amino group at the side chain of the substrate C-terminus. On the other hand, CPB is similar to trypsin (ibid) in that the best substrate would have a side chain length between those of lysine and arginine.Studies with Bz-MeGly-Lys and Bz-Ala-Lys showed that the former is very slowly hydrolyzed by CPB but that the latter is a good substrate, with a high affinity for the enzyme, indicative of considerable participation of the Cα-methyl group of alanine in the binding of the substrate to the enzyme.
Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid.
