Secondary interactions in mesentericopeptidase-catalyzed hydrolysis of peptide ester and 4-nitroanilide substrates

Abstract Five substrate series with the formulae Z-(Gly) n -Phe-OMe, Z-(Ala) n -Phe-OMe, Ac-(Ala) n -Phe-OMe, Z-(Gly) n -Phe-NA, and Suc-(Gly) n -Phe-NA ( n = 0–2) (Z-benzyloxy-carbonyl) were synthesized and used to study the active site of mesentericopeptidase (EC 3.4.21). The elongation of the peptide chain in all series leads to a 100- to 300-fold increase of k cat K m . This indicates an extended substrate binding site, comprising at least three subsites (S 1 -S 3 ). The sequence P 1 -P 3 that fits these subsites is PheAlaAla. Mesentericopeptidase responds to the elongation of the peptide chain in the series Ac-(Ala) n -Phe-OMe in a way similar, but not identical, to subtilisin Carlsberg and subtilisin BPN′. The poor amidolytic activity of mesentericopeptidase and subtilisins toward 4-nitroanilides with peptide sequences matching the S 1 -S 3 subsites is discussed in terms of unfavorable S′ 1 -P′ 1 interaction.