Action of Carboxypeptidase on Synthetic Substrates

Abstract

The rates of hydrolysis of synthetic substrates by CPase depend chiefly on the nature of the C-terminal residue, and acyl dipeptide analogs containing aromatic amino acid and leucine of the L-configuration in the C-terminal position fulfil optimal structural requirements; CbzGly-L-Phe is most sensitive among substrates known for the enzyme (1). Although the side chain specificity of the amino acids forming the susceptible bond is a dominant factor, it should be mentioned that the rate of release of C-terminal amino acid from a protein may also be influenced by the environmental conditions imposed on the sensitive bond by virtue of the size and spacial configuration of polypeptide chains in the molecule. In this connection, it seemed desirable to examine the mode and rates of hydrolysis of the peptides Gly_n-L-Tyr by CPase in which n are 1, 2, 3, 4 and 5. Moreover, a study designed in this paper appeared valuable in view of the widespread use of the enzyme to determine C-terminal residues and amino acid sequences of proteins and peptides (2). Earlier studies have shown that CPase hydrolyzes, though at low rates, the dipeptides such as Gly-L-Phe (3), Ala-3-Phe (3), Gly-L-Tyr (4, 5), Gly-L-Try (5), Gly-L-Leu (5), L-Tyr-L-Tyr (4) and Leu-L-Tyr (5). However, the introduction of glycyl residue to N-terminal position of L-Leu-L-Tyr resulted in remarkable increase in hydrolytic rate; Gly-L-Leu-L-Tyr was hydrolyzed 38 times more rapidly than L-Leu-L-Tyr (3)Plentl and Page have shown that the terminal peptide bond of the tetrapeptide L-Tyr-L-Lys-L-Glu-L-Tyr was susceptible to hydrolysis by CPase (6). Yanari and Mitz have shown that L-LeuGlyGly and L-LeuGly are resistant to hydrolysis even with relatively large amount of CPase (5). In the present investigation, it was shown by means of paper chromatography that the peptides Gly_n-L-Tyr (n=2∼5) are hydrolyzed to corresponding Gly_n (n = 2∼5) and L-tyrosine by CPase, the polyglycine Gly_n (n = 2∼5) being not split appreciably further. In the synthesis of the substrates tested, the mixed anhydrides of CbzGly_n (n=l∼4) were coupled with L-tyrosine benzyl ester or glycyl-L-tyrosine benzyl ester by the procedure of Vaughan and Osato (7), and after hydrogenation of the coupling products, the peptides Glyn-L-Tyr (n=l∼5) were obtained.