Organelle and translocatable forms of glyoxysomal malate dehydrogenase - The effect of the N-terminal presequence
Organelle and translocatable forms of glyoxysomal malate dehydrogenase - The effect of the N-terminal presequence
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DOI：
10.1111/j.1742-4658.2004.04475.x
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发表时间：
2005-02-01
期刊：
影响因子：
5.4
通讯作者：
Banaszak, L
中科院分区：
文献类型：
--
作者：
Cox, B;Chit, MM;Banaszak, L
许多由核基因编码的细胞器酶具有N末端序列，其引导它们进入细胞器（前体）并在输入时被移除（成熟）。下面描述的实验表征西瓜乙醛酸脱氢酶的前体和成熟形式之间的差异。使用重组蛋白方法，使用Ni 2 +-NTA亲和层析将前体（p-gMDH）和成熟（gMDH）形式纯化至均一。凝胶过滤和动态光散射已经显示gMDH和p-gMDH在溶液中是二聚体，其中p-gMDH具有相应更高的分子量。p-gMDH在4 - 32 ℃之间的温度下也表现出较小的平移扩散系数（D-t），这是由于N-末端上的额外氨基酸造成的。差示扫描量热法描述了两种蛋白质的解折叠性质的显著差异，其中p-gMDH显示出额外的温度依赖性转变。此外，草酰乙酸的稳态动力学常数和K-m的pH依赖性中发现了一些差异。这两个细胞器的前体和成熟形式的这种glyoxysomal酶在相同的条件下结晶。p-gMDH的晶体结构（可切割和可转位蛋白质的第一结构）被解析至2.55埃的分辨率。GMDH是第一个乙醛酶体MDH结构，并被解析到2.50埃的分辨率。两种结构的比较表明，在p-gMDH、gMDH和其它MDH的相应元件之间几乎没有可见的三级或四级结构差异。来自成熟蛋白和可转位蛋白的图谱在G44之前缺乏显著的电子密度。虽然在生物二聚体中没有来自任一单体的易位序列的部分可见，但所有其他溶液性质表明在N-末端的额外残基的可测量的影响。
Many organelle enzymes coded for by nuclear genes have N-terminal sequences, which directs them into the organelle (precursor) and are removed upon import (mature). The experiments described below characterize the differences between the precursor and mature forms of watermelon glyoxysomal malate dehydrogenase. Using recombinant protein methods, the precursor (p-gMDH) and mature (gMDH) forms were purified to homogeneity using Ni2+-NTA affinity chromatography. Gel filtration and dynamic light scattering have shown both gMDH and p-gMDH to be dimers in solution with p-gMDH having a correspondingly higher molecular weight. p-gMDH also exhibited a smaller translational diffusion coefficient (D-t) at temperatures between 4 and 32 degreesC resulting from the extra amino acids on the N-terminal. Differential scanning calorimetry described marked differences in the unfolding properties of the two proteins with p-gMDH showing additional temperature dependent transitions. In addition, some differences were found in the steady state kinetic constants and the pH dependence of the K-m for oxaloacetate. Both the organelle-precursor and the mature form of this glyoxysomal enzyme were crystallized under identical conditions. The crystal structure of p-gMDH, the first structure of a cleavable and translocatable protein, was solved to a resolution of 2.55 Angstrom. GMDH is the first glyoxysomal MDH structure and was solved to a resolution of 2.50 Angstrom. A comparison of the two structures shows that there are few visible tertiary or quaternary structural differences between corresponding elements of p-gMDH, gMDH and other MDHs. Maps from both the mature and translocatable proteins lack significant electron density prior to G44. While no portion of the translocation sequences from either monomer in the biological dimer was visible, all of the other solution properties indicated measurable effects of the additional residues at the N-terminal.