Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912.
Plant Physiol. 1988 Nov;88(3):879-86. doi: 10.1104/pp.88.3.879.
The universal tetrapyrrole precursor delta-aminolevulinic acid (ALA) is formed from glutamate (Glu) in algae and higher plants. In the postulated reaction sequence, Glu-tRNA is produced by a Glu-tRNA synthetase, and the product serves as a substrate for a reduction step catalyzed by a pyridine nucleotide-requiring Glu-tRNA dehydrogenase. The reduced intermediate is then converted into ALA by a transaminase. An RNA and three enzyme fractions required for ALA formation from Glu have been isolated from soluble Chlorella extracts. The recombined fractions catalyzed ALA production from Glu or Glu-tRNA. The fraction containing the synthetase produced Glu-tRNA from Glu and tRNA in the presence of ATP and Mg(2+). The isolated product of this reaction served as substrate for ALA production by the partially reconstituted enzyme system lacking the synthetase fraction and incapable of producing ALA from Glu. The production of ALA from Glu-tRNA by this partially reconstituted system did not require free Glu or ATP, and was not affected by added ATP. These results show that (a) free Glu-tRNA is an intermediate in the formation of ALA from Glu, (b) ATP is required only in the first step of the reaction sequence, and NADPH only in a later step, (c) Glu-tRNA production is the essential reaction catalyzed by one of the enzyme fractions, (d) this enzyme fraction is active in the absence of the other enzymes and is not required for activity of the others. The specific Glu-tRNA synthetase required for ALA formation has an approximate molecular weight of 73,000 +/- 5,000 as determined by Sephadex G-100 gel filtration and native polyacrylamide gel electrophoresis. Other Glu-tRNA synthetases were present in the cell extracts but were ineffective in the the ALA-forming process.
普遍的四吡咯前体δ-氨基乙酰丙酸（ALA）是在藻类和高等植物中由谷氨酸（Glu）形成的。在假定的反应序列中，Glu-tRNA 由 Glu-tRNA 合成酶产生，产物作为需要吡啶核苷酸的 Glu-tRNA 脱氢酶催化的还原步骤的底物。然后，还原中间体通过转氨基酶转化为 ALA。已从可溶的小球藻提取物中分离出用于从 Glu 形成 ALA 的 RNA 和三种酶级分。重组级分催化 Glu 或 Glu-tRNA 生成 ALA。含有合成酶的级分从 Glu 和 tRNA 产生 Glu-tRNA，在 ATP 和 Mg(2+)存在的情况下。该反应的分离产物作为缺少合成酶级分且不能从 Glu 产生 ALA 的部分重组酶系统的 ALA 产生的底物。该部分重组系统从 Glu-tRNA 产生 ALA 不需要游离的 Glu 或 ATP，并且不受添加的 ATP 的影响。这些结果表明：（a）游离的 Glu-tRNA 是 Glu 形成 ALA 的中间产物，（b）仅在反应序列的第一步中需要 ATP，仅在稍后的步骤中需要 NADPH，（c）Glu-tRNA 的产生是一种酶级分催化的必需反应，（d）该酶级分在没有其他酶的情况下具有活性，并且不需要其他酶的活性。所需的特定的 Glu-tRNA 合成酶用于 ALA 形成，其分子量约为 73,000 +/- 5,000，如通过 Sephadex G-100 凝胶过滤和天然聚丙烯酰胺凝胶电泳确定的。细胞提取物中存在其他的 Glu-tRNA 合成酶，但在 ALA 形成过程中无效。
