Effect of mutations at Met-88 and Met-90 on the biotination of Lys-89 of the apo 1.3S subunit of transcarboxylase.
Effect of mutations at Met-88 and Met-90 on the biotination of Lys-89 of the apo 1.3S subunit of transcarboxylase.
复制标题
Met-88 和 Met-90 突变对转羧酶 apo 1.3S 亚基 Lys-89 生物素化的影响。
DOI：
10.1096/fasebj.2.9.3131174
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Wood,HG
中科院分区：
文献类型：
--
作者：
Shenoy,BC;Paranjape,S;Murtif,VL;Kumar,GK;Samols,D;Wood,HG
转羧酶的 apo 1.3S 亚基包含序列 Ala-87-Met-88-Lys-89-Met-90，其中 Lys-89 是生物素化的。该序列在所有已测序的生物素酶中高度保守（鸡肝中的乙酰辅酶A羧化酶除外，该酶用 Val 代替了 Ala）。通过定点诱变检查了 Met-88 和 Met-90 在合成酶对 Lys-89 进行生物素化时的作用。通过寡核苷酸定向体外诱变产生编码 Thr-88、Leu-88 或 Leu-90 的 1.3S 亚基基因，并在大肠杆菌中表达。分离突变的 apo 1.3S 亚基，并将谢尔曼丙酸杆菌均质合成酶的生物素化与 apo 野生型亚基的生物素化进行比较。 apo突变体的Vmax与apo野生型相同，但当Leu取代Met-88或Met-90时，突变体的Km低于野生型或突变体Thr-88。 E.合成酶的活性。通过体内测定测定大肠杆菌。在对数生长早期，与野生型或突变体 Leu-88 相比，突变体 Thr-88 和 Leu-90 的一小部分被生物素化。当培养物进入稳定期时，突变体和野生型被生物素化至相同程度。总体结果表明，合成酶对 apo 1.3S 亚基进行生物素化不需要 Met-88 和 Met-90。-Shenoy，B. C.；帕兰贾普，S.；穆蒂夫，V.L.；库马尔，G.K.；萨莫尔斯，D； Wood, H. G. Met-88 和 Met-90 突变对转羧酶 apo 1.3S 亚基 Lys-89 生物素化的影响。FASEB J.2：2505-2511； 1988年。
The apo 1.3S subunit of transcarboxylase contains the sequence Ala‐87‐Met‐88‐Lys‐89‐Met‐90, and it is Lys‐89 that is biotinated. This sequence is highly conserved in all the biotin enzymes that have been sequenced (with the exception of acetyl‐CoA carboxylase from chicken liver, which has Val in place of Ala). The role of Met‐88 and Met‐90 in specifying Lys‐89 for biotination by synthetase was examined by site‐directed mutagenesis. Genes of the 1.3S subunit coding for Thr‐88, Leu‐88, or Leu‐90 were generated by oligonucleotide‐directed in vitro mutagenesis and expressed inEscherichia coli.The mutated apo 1.3S subunits were isolated and the biotination by homogeneous synthetase fromPropionibacterium shermaniiwas compared with that of the apo wild‐type subunit. TheVmaxfor the apo mutants was the same as that for the apo wild type, but when Leu was substituted for Met‐88 or Met‐90, theKmfor the mutant was lower than that of the wild‐type or mutant Thr‐88. The activity of the synthetase ofE. coliwas determined by an in vivo assay. During the early log phase of growth, a smaller portion of mutants Thr‐88 and Leu‐90 was biotinated than with the wild‐type or mutant Leu‐88. When the cultures progressed to stationary phase, mutants and the wild type were biotinated to the same extent. The overall results show that Met‐88 and Met‐90 are not required for biotination of the apo 1.3S subunit by the synthetases.—Shenoy, B. C.; Paranjape, S.; Murtif, V. L.; Kumar, G. K.; Samols, D; Wood, H. G. Effect of mutations at Met‐88 and Met‐90 on the biotination of Lys‐89 of the apo 1.3S subunit of transcarboxylase.FASEB J.2: 2505‐2511; 1988.