Brems D N, Bruenger E, Rilling H C
Biochemistry. 1981 Jun 23;20(13):3711-8. doi: 10.1021/bi00516a007.
Previously we presented evidence for the selective modification of the catalytic site of prenyltransferase by photoaffinity labeling with o-azidophenylethyl pyrophosphate [Brems, D. N., & Rilling, H. C. (1979) Biochemistry 18, 860]. In the present work, we report the isolation and characterization of a CNBr fragment of 30 amino acid residues from the photoaffinity-labeled enzyme. This CNBr fragment contains over 809% of the total label attached to prenyltransferase as a result of photoaffinity labeling. Several lines of evidence indicate that a number of residues in this CNBr fragment have been modified. First, Edman degradation of this labeled peptide demonstrates that at least 16 of the 30 amino acids have been modified by the photoaffinity reagent. The two most extensively modified amino acids are a specific arginine and alanine. Second, two-dimensional chromatography of Pronase digestions of the labeled CNBr fragment indicates that at least 11 different products resulted from photoaffinity labeling. Third, peptide maps of a trypsin digest of this CNBr fragment show that the attached affinity label is distributed among at least three of the resulting products of tryptic hydrolysis. Finally, comparison of amino acid analysis of this CNBr fragment with that of its counterpart isolated from native enzyme is consistent with the modification of a number of amino acids rather than a few y the photoaffinity labeling process.
此前我们通过用邻叠氮苯乙基焦磷酸进行光亲和标记，提供了异戊烯基转移酶催化位点被选择性修饰的证据[布雷姆斯，D. N.，& 里林，H. C.（1979年）《生物化学》18，860]。在本研究中，我们报告了从光亲和标记的酶中分离并鉴定出一个含30个氨基酸残基的溴化氰片段。这个溴化氰片段含有因光亲和标记而附着在异戊烯基转移酶上的总标记的80%以上。多条证据表明该溴化氰片段中的一些残基已被修饰。首先，对这个标记肽进行埃德曼降解表明，30个氨基酸中至少有16个已被光亲和试剂修饰。修饰程度最高的两个氨基酸是一个特定的精氨酸和丙氨酸。其次，对标记的溴化氰片段进行链霉蛋白酶消化后的二维色谱分析表明，光亲和标记产生了至少11种不同的产物。第三，该溴化氰片段胰蛋白酶消化产物的肽图显示，附着的亲和标记分布在胰蛋白酶水解产生的至少三种产物中。最后，将这个溴化氰片段的氨基酸分析与其从天然酶中分离出的对应片段进行比较，结果与光亲和标记过程中多个氨基酸而非少数氨基酸被修饰的情况一致。
