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.

各种蛋白质中天冬氨酰和天冬酰胺基残基的结构改变可能导致其功能失常，从而可能导致严重的健康失调。琥珀酰亚胺中间体的形成和水解对于特定的蛋白质修饰至关重要。尽管如此，只有很少的研究调查琥珀酰亚胺中间体的水解。在这项研究中，我们建立了一种使用重组蛋白I制备带有琥珀酰亚胺基残基的肽的方法-异天冬氨酰甲基转移酶和超滤单元。使用琥珀酰亚胺基肽，我们检查了氨基酸残基对琥珀酰亚胺基残基羧基端的琥珀酰亚胺基水解的影响，并确定了每种肽的水解速率常数。羧基端带有Ser残基的肽中的琥珀酰亚胺水解速率常数（0.50±0.02 / h）比带有Ala残基的肽（0.17±0.01 / h）高3.0倍，而仅为1.2带有Gly残基的肽（0.20±0.01 / h）的高倍数 带有Ser残基的肽[（2.44±0.11）×10 ^-3 / d]的琥珀酰亚胺形成速率常数仅比带有Ala残基的肽（[1.87±0.09）×10 ^-3的高1.2倍。/ d]，而带有Gly残基的肽要高5.5倍[（10.2±0.2）×10 ^-3 / d]。这些结果表明，在琥珀酰亚胺基残基的羧基末端的Gly和Ser残基在琥珀酰亚胺基的形成和水解中具有相反的作用。Ser残基的羟基催化在琥珀酰亚胺水解中起关键作用。