Galletti P, Ciardiello A, Ingrosso D, Di Donato A, D'Alessio G
Istituto di Biochimica delle Macromolecole, I Facoltà di Medicina e Chirurgia, Università di Napoli, Italy.
Biochemistry. 1988 Mar 8;27(5):1752-7. doi: 10.1021/bi00405a055.
Previous work has shown that in the peptide segment 62-76 of naturally deamidated alpha subunit of bovine seminal ribonuclease (BS-RNase) the alpha-carboxyl group of iso-Asp67 is selectively methylated by S-adenosylmethionine:protein carboxyl O-methyltransferase [Di Donato, A., Galletti, P., & D'Alessio, G. (1986) Biochemistry 25, 8361-8368]. In the present study this reaction has been characterized, by using the tryptic segment 62-76 of the protein chain (peptide alpha 16). The peptide is stoichiometrically methyl esterified with a Km of 6.17 microM and a Vmax of 19.56 nmol min-1 mg-1, and the product of demethylation has been identified as the cyclic succinimidyl derivative of iso-Asp67-Gly68. The cleavage of the succinimidyl ring yields two isomeric peptides containing an aspartyl residue (peptide alpha 17) and an isoaspartyl residue (peptide alpha 16). On the basis of these results conditions were defined in which repeated cycles of methylation-demethylation led to an effective conversion of peptide alpha 16 into peptide alpha 17, a process that can be interpreted as the repair of an altered isopeptide bond. When the methyl esterification reaction was studied on the native dimeric isoenzymes of seminal RNase and on catalytically active monomeric derivatives, including a stabilized alpha-type subunit, the results of these experiments showed that none of the protein forms were substrates for the methyltransferase. Only the unfolded alpha-type subunit was methylated to a stoichiometric extent. These results indicate that the repair of altered isopeptide bonds is chemically feasible in peptides but is hindered in the case of seminal RNase by its three-dimensional structure.
先前的研究表明，在牛精核糖核酸酶（BS-RNase）天然脱酰胺化α亚基的62-76肽段中，异天冬氨酸67（iso-Asp67）的α-羧基可被S-腺苷甲硫氨酸：蛋白质羧基O-甲基转移酶选择性甲基化[迪多纳托，A.，加莱蒂，P.，& 达莱西奥，G.（1986年）《生物化学》25卷，8361 - 8368页]。在本研究中，通过使用蛋白质链的胰蛋白酶消化片段62-76（肽α16）对该反应进行了表征。该肽以化学计量比甲基酯化，米氏常数（Km）为6.17微摩尔，最大反应速度（Vmax）为19.56纳摩尔每分钟每毫克，去甲基化产物已被鉴定为异天冬氨酸67-甘氨酸68（iso-Asp67-Gly68）的环状琥珀酰亚胺衍生物。琥珀酰亚胺环的裂解产生两种含有天冬氨酸残基的异构肽（肽α17）和一种异天冬氨酸残基的肽（肽α16）。基于这些结果，确定了重复甲基化-去甲基化循环导致肽α16有效转化为肽α17的条件，该过程可解释为对改变的异肽键的修复。当对精核糖核酸酶的天然二聚体同工酶以及包括稳定化α型亚基在内的催化活性单体衍生物进行甲酯化反应研究时，这些实验结果表明，这些蛋白质形式均不是甲基转移酶的底物。只有未折叠的α型亚基被甲基化至化学计量水平。这些结果表明，改变的异肽键的修复在肽中在化学上是可行的，但在精核糖核酸酶的情况下，由于其三维结构而受到阻碍。
