Identification by mutagenesis of arginines in the substrate binding site of the porcine NADP-dependent isocitrate dehydrogenase
Identification by mutagenesis of arginines in the substrate binding site of the porcine NADP-dependent isocitrate dehydrogenase
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DOI：
10.1074/jbc.275.8.5606
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发表时间：
2000-02-25
影响因子：
4.8
通讯作者：
Colman, RF
中科院分区：
文献类型：
--
作者：
Soundar, S;Danek, BL;Colman, RF
Pig Heart线粒体NADP依赖性异位酸脱氢酶是在哺乳动物异质脱氢酶中研究最广泛的研究。大肠杆菌脱氢酶和大肠杆菌异位酸脱氢酶的晶体结构和效率比对的序列比对，这表明猪ARG（101），ARG（110），ARG（120），ARG（120）和ARG（133）是在底物结合的孔中的候选者。使用聚合酶链反应方法将四种精氨酸分别突变为谷氨酰胺。野生型和突变酶均在大肠杆菌中分离为麦芽糖结合融合蛋白，然后用凝血酶裂解，并纯化以产生同质性猪异西酸脱氢酶。 R120Q突变体具有特定活性，以及类似于野生型酶的等二酸，MN2+和NADP（+）的K-M值，表明功能不需要ARG（120）。 R101Q，R110Q和R133Q的特定活性分别为1.73、1.30和19.7 Mu mols/min/mg，而野生型酶则为39.6单位/mg。与野生型相比，R110Q和R133Q酶的异位酸盐的K-M值分别增加了400倍和165倍以上。 MN2+的K-M值，但对于NADP（+）而言，也升高，表明金属 - 异构酸酯复合物的结合在这些突变体中受损。有人提出，ARG（110）和ARG（133）的阳性电荷通常通过静电吸引力加强带负电荷的异位酸的结合。 R101q突变体显示出较小但同二+的K-M值显着增加。但是，K（CAT）的明显下降表明ARG（101）在催化中的作用。野生型酶的V-Max取决于PK 5.5的酶基的电离形式，而该PK（AES）相似，用于R101Q和R120Q酶。相反，R110Q和R133Q酶的PK（AES）分别增加到6.4和7.4，表明ARG（110）和ARG（133）的正电荷通常降低附近催化碱基的PK以促进其电离。这些结果可以从基于大肠杆菌酶的X射线坐标产生的猪NADP特异性脱氢酶的结构来理解。
Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The crystal structure of Escherichia cell isocitrate dehydpogenase and sequence alignment of porcine with E. coli isocitrate dehydrogenase suggests that the porcine Arg(101), Arg(110), Arg(120), and Arg(133) are candidates for roles in substrate binding. The four arginines were separately mutated to glutamine using a polymerase chain reaction method. Wild type and mutant enzymes were each expressed in E. coli, isolated as maltose binding fusion proteins, then cleaved with thrombin, and purified to yield homogeneous porcine isocitrate dehydrogenase. The R120Q mutant has a specific activity, as well as K-m values for isocitrate, Mn2+, and NADP(+) similar to wild type enzyme, indicating that Arg(120) is not needed for function. The specific activities of R101Q, R110Q, and R133Q are 1.73, 1.30, and 19.7 mu mols/min/mg, respectively, as compared with 39.6 units/mg for wild type enzyme. The R110Q and R133Q enzymes exhibit K-m values for isocitrate that are increased more than 400- and 165-fold, respectively, as compared with wild type. The K-m values for Mn2+, but not for NADP(+), are also elevated indicating that binding of the metal-isocitrate complex is impaired in these mutants. It is proposed that the positive charges of Arg(110) and Arg(133) normally strengthen the binding of the negatively charged isocitrate by electrostatic attraction. The R101Q mutant shows smaller, but significant increases in the K-m values for isocitrate and Mn2+; however, the marked decrease in k(cat) suggests a role for Arg(101) in catalysis The V-max of wild type enzyme depends on the ionized form of an enzymic group of pK 5.5, and this pK(aes) is similar for the R101Q and R120Q enzymes. In contrast, the pK(aes) for R110Q and R133Q enzymes increases to 6.4 and 7.4, respectively, indicating that the positive charges of Arg(110) and Arg(133) normally lower the pK of the nearby catalytic base to facilitate its ionization. These results may be understood in terms of the structure of the porcine NADP-specific isocitrate dehydrogenase generated by the Insight Modeler Program, based on the x-ray coordinates of the E. coli enzyme.