Isolation and sequencing of an active-site peptide from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase after affinity labeling with 2-[(bromoacetyl)amino]pentitol 1,5-bisphosphate.
Isolation and sequencing of an active-site peptide from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase after affinity labeling with 2-[(bromoacetyl)amino]pentitol 1,5-bisphosphate.
复制标题
用 2-[(溴乙酰基)氨基]戊醇 1,5-二磷酸亲和标记后，对红色红螺菌二磷酸核酮糖羧化酶/加氧酶的活性位点肽进行分离和测序。
DOI：
10.1021/bi00275a028
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发表时间：
1983
期刊：
影响因子：
2.9
通讯作者：
F. C. Hartman
中科院分区：
文献类型：
--
作者：
B. Fraij;F. C. Hartman
据报道，2-[（溴乙酰基）氨基]戊糖醇1，5-二磷酸是来自红杜鹃的核酮糖二磷酸羧化酶/加氧酶的高选择性亲和标记[Fraij，B.，& Hartman，F. C.（1982）J.Biol.Chem.257，3501-3505]。现在已经用14 C标记的试剂灭活了酶，以便在序列水平上鉴定靶残基。失活后，用碘乙酸羧甲基化酶，然后用胰蛋白酶消化。通过在DEAE-纤维素、SP-Sephadex和Sephadex G-25上连续层析，以高纯度获得消化物中唯一的放射性肽。基于纯化肽的氨基酸分析，衍生的残余物是甲硫氨酰锍盐。自动Edman降解证实了标记肽的纯度，并确定其序列为Leu-Gln- Gly-Ala-Ser-Gly-Ile-His-Thr-Gly-Thr-Met-Gly-Phe-Gly-Lys-Met-Glu-Gly-Glu-Ser-Ser - Asp-Arg。用溴化氰切割该肽显示试剂部分共价连接到第二个甲硫氨酰残基。序列同源性与羧化酶/加氧酶从菠菜表明，赖氨酰残基之前立即烷基化蛋氨酸对应于赖氨酸-334，一个残基先前牵连在活性位点。
2-[(Bromoacetyl)amino]pentitol 1,5-bisphosphate was reported to be a highly selective affinity label for ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum [Fraij, B., & Hartman, F. C. (1982) J. Biol. Chem. 257, 3501-3505]. The enzyme has now been inactivated with a 14C-labeled reagent in order to identify the target residue at the sequence level. Subsequent to inactivation, the enzyme was carboxymethylated with iodoacetate and then digested with trypsin. The only radioactive peptide in the digest was obtained at a high degree of purity by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. On the basis of amino acid analysis of the purified peptide, the derivatized residue was a methionyl sulfonium salt. Automated Edman degradation confirmed the purity of the labeled peptide and established its sequence as Leu-Gln- Gly-Ala-Ser-Gly-Ile-His-Thr-Gly-Thr-Met-Gly-Phe-Gly-Lys-Met-Glu-Gly-Glu-Ser-Ser - Asp-Arg. Cleavage of this peptide with cyanogen bromide showed that the reagent moiety was covalently attached to the second methionyl residue. Sequence homology with the carboxylase/oxygenase from spinach indicates that the lysyl residue immediately preceding the alkylated methionine corresponds to Lys-334, a residue previously implicated at the active site.