STUDIES ON ACTIVE SITE OF ENZYME RIBULOSE-DIPHOSPHATE CARBOXYLASE
STUDIES ON ACTIVE SITE OF ENZYME RIBULOSE-DIPHOSPHATE CARBOXYLASE
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DOI：
10.1016/0005-2744(67)90166-0
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发表时间：
1967-01-01
期刊：
影响因子：
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通讯作者：
METHENITOU, H
中科院分区：
文献类型：
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作者：
AKOYUNOGLOU, G;ARGYROUD.JH;METHENITOU, H
磷酸核丁酮羧化酶（3-磷酸- d -甘油羧化酶（二聚），EC 4.1.1.39，以前称为羧歧化酶），是光合作用中催化二氧化碳固定反应的酶，与二氧化碳形成络合物，可以通过离子交换柱分离。通过用重氮甲烷稳定C14O2-酶复合物，用胰蛋白酶（EC 3.4.4.4）消化水解，离子交换柱层析分离所得肽，可以得到高放射性肽。此外，分离的放射性肽呈微色，这就提出了关于发色团的性质和功能的问题。放射性肽可被胃蛋白酶（EC 3.4.4.1）或羧肽酶（EC 3.4.2.1）进一步水解。离子交换柱层析法分离胃蛋白酶水解产物，只得到一个放射性部分，该部分也着色。该组分的紫外吸收光谱很大程度上取决于溶液的pH值。羧肽酶水解产物经纸层析得到1个放射性点和14个茚三酮阳性点。用c14 -标记-氨基乙酰丙酸进行的实验表明，分离磷酸核酮糖羧化酶的色氨酸肽后，彩色肽中存在放射性。
Ribulosediphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39, formerly known as carboxydismutase), the enzyme catalyzing the CO2 fixation reaction in photosynthesis, forms a complex with CO2 that can be isolated by passage through an ion-exchange column. By using diazomethane to stabilize the C14O2- enzyme complex, digestion with trypsin (EC 3.4.4.4) to hydrolyze it, and ion-exchange column chromatography to separate the resulting peptides, it has been possible to obtain a highly radioactive peptide. Moreover, the isolated radioactive peptide is slightly colored, which raises the question as to the nature and function of the chromophore. The radioactive peptide can be further hydrolyzed by pepsin (EC 3.4.4.1) or carboxypeptidase (EC 3.4.2.1). Separation of the pepsin hydrolysate by ion-exchange column chromatography gave only one radioactive fraction, which was also colored. The ultraviolet absorption spectrum of this fraction depends greatly on the pH of the solution. Resolution of the carboxypeptidase hydrolysate by paper chromatography gave one radioactive spot and 14 ninhydrin-positive spots. Experiments with C14-iabeleds -aminolevulinic acid showed that after separation of the tryptic peptides of ribulosediphosphate carboxylase, radioactivity was present in the colored peptide.