Effect of N-methylation and chain length on kinetic constants of trypsin substrates. ε-N-Methyllysine and homolysine derivatives as substrates

Abstract

The action of trypsin on the following amino acid derivatives has been investigated: the ethyl esters of ε-N-mono-, ε-N-di-, and ε-N-tri-methyl-L-lysine; the ethyl esters of the homologues of lysine and arginine; the methyl ester and amide of the α-N-benzoyl-DL-homolysine; the methyl esters and amides of the α-N-benzoyl derivatives of ε-N-di- and ε-N-tri-methyllysine; and poly ε-N-methyllysine. Derivatives of L-ornithine, DL-2,8-diaminooctanoic acid, ε-N-dimethyl-, ε-N-trimethyl-, and ε-N-formyl-L-lysine were not substrates of trypsin. ε-N-Dimethyl-L-lysine derivatives did not inhibit the action of trypsin on a specific substrate. DL-Homolysine derivatives were hydrolyzed with kcat's one to two orders of magnitude lower than those of lysine derivatives, but their Km's were only 1.5–3 times higher. ε-N-Methyl-L-lysine derivatives were hydrolyzed at rates similar to those for DL-homolysine derivatives, and had Km's 25–115 times those of lysine derivatives. Plots of Km and kcat/Km versus side-chain length of the substrate for the ethyl esters of all the homologues of lysine and arginine indicated a correlation between these kinetic constants and side-chain length, and that the best substrate would have a side-chain length between those of lysine and arginine. Poly-ε-N-methyl-L-lysine was degraded to small peptides by trypsin.