Effect of amino acids present at the carboxyl end of succinimidyl residue on the rate constants for succinimidyl hydrolysis in small peptides
2020
Sadakane, Yutaka | Senda, Sayumi | Deguchi, Taku | Tanaka, Atsuki | Tsuruta, Hiromasa | Morimoto, Shota
Structural alterations of aspartyl and asparaginyl residues in various proteins can lead to their malfunction, which may result in severe health disorders. The formation and hydrolysis of succinimidyl intermediates are crucial in specific protein modifications. Nonetheless, only few studies investigating the hydrolysis of succinimidyl intermediates have been published. In this study, we established a method to prepare peptides bearing succinimidyl residues using recombinant protein l-isoaspartyl methyltransferase and ultrafiltration units. Using succinimidyl peptides, we examined the effect of amino acid residues on succinimidyl hydrolysis at the carboxyl end of succinimidyl residues and determined the rate constant of hydrolysis for each peptide. The rate constant of succinimidyl hydrolysis in the peptide bearing a Ser residue at the carboxyl side (0.50 ± 0.02 /h) was 3.0 times higher than that for the peptide bearing an Ala residue (0.17 ± 0.01 /h), whereas it was just 1.2 times higher for the peptide bearing a Gly residue (0.20 ± 0.01 /h). The rate constant of succinimidyl formation in the peptide bearing a Ser residue [(2.44 ± 0.11) × 10⁻³ /d] was only 1.2 times higher than that for the peptide bearing an Ala residue ([1.87 ± 0.09) × 10⁻³ /d], whereas 5.5 times higher for the peptide bearing a Gly residue [(10.2 ± 0.2) × 10⁻³ /d]. These results show that the Gly and Ser residues at the carboxyl end of the succinimidyl residue have opposing roles in succinimidyl formation and hydrolysis. Catalysis of Ser residue's hydroxyl group plays a crucial role in succinimidyl hydrolysis.
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