Characterization of the carboxylate delivery module of transcarboxylase: following spontaneous decarboxylation of the 1.3S-CO2- subunit by NMR and FTIR spectroscopies.
Characterization of the carboxylate delivery module of transcarboxylase: following spontaneous decarboxylation of the 1.3S-CO2- subunit by NMR and FTIR spectroscopies.
复制标题
转羧酶的羧酸盐递送模块的表征：通过 NMR 和 FTIR 光谱对 1.3S-CO2- 亚基自发脱羧。
DOI：
10.1021/bi0116442
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Carey,PaulR
中科院分区：
文献类型：
--
作者：
Rivera-Hainaj,RosaE;Pusztai-Carey,Marianne;VenkatReddy,D;Choowongkomon,Kiattawee;Sönnichsen,FrankD;Carey,PaulR
转羧酶（TC）是一种多亚基酶，催化羧酸基团从甲基丙二酰辅酶A（MMCoA）转移到丙酮酸。CO2-基团通过与1.3S亚基共价连接的生物素辅因子在MMCoA和丙酮酸结合位点之间穿梭。完全羧化的1.3S可以在体外使用1.3S、MMCoA和催化量的TC的MMCoA结合亚基制备。1.3S−CO2中间体在数小时内自发地脱羧，这个过程通过1D和2D NMR和FTIR光谱进行了表征。核磁共振数据得到自发脱羧反应的一级动力学常数为1.4 × 10- 3 min-1。通过测量生物素的脲基NH质子的再现和来自1.3S的N-末端的归属于Asn-8和/或Asn-24的6.99和7.67 ppm处的峰的消失，从1D NMR光谱计算该速率。由于N末端内两种或更多种构象之间的交换导致谱线增宽，非羧化1.3S的1D谱中不存在后一个峰。有人提出，生物素-CO2-和N-末端氨基酸之间的相互作用扰乱这种构象平衡，导致一些N-末端残基出现在1D NMR谱的羧化形式。通过比较1. 3S-CO2-和1.3S蛋白质的二维光谱，进一步的细节是显而易见的，其中羧基化导致C-末端一半的几个峰发生位移，以及由于位于蛋白质N-末端一半的一些残基而出现共振。FTIR差谱也用于跟踪1.3S−CO2-的自发脱羧。对于羧基化的1.3S，差谱提供了生物素环上的CO2-的振动特征。在1695和1699 cm-1处发现了一个双峰，其强度随t的增加而增加。这被归因于与1.3S蛋白上的生物素结合的CO2-基团的反对称伸缩振动。它的位置和轮廓为生物素-CO2-基团和1.3S蛋白之间发生的相互作用提供了进一步的证据。这些研究证明了1.3S蛋白质的高度移动的、“稳定的”性质，该蛋白质因其作为CO2转运蛋白的作用而被工程化。
Transcarboxylase (TC) is a multisubunit enzyme that catalyzes the transfer of a carboxylate group from methylmalonyl-CoA (MMCoA) to pyruvate. The CO2-group is shuttled between the MMCoA and pyruvate binding sites by a biotin cofactor, covalently linked to the 1.3S subunit. Fully carboxylated 1.3S can be prepared in vitro using 1.3S, MMCoA, and catalytic amounts of the TC's MMCoA-binding subunit. The 1.3S−CO2-intermediate decarboxylates spontaneously over a period of hours, and this process was characterized by 1D and 2D NMR and FTIR spectroscopies. The NMR data yielded a first-order kinetic constant of 1.4 × 10-3min-1for the spontaneous decarboxylation. This rate was calculated from the 1D NMR spectrum by measuring the reappearance of biotin's ureido NH protons and the disappearance of peaks at 6.99 and 7.67 ppm assigned to Asn-8 and/or Asn-24 from the 1.3S's N-terminus. The latter peaks are absent in the 1D spectrum of non-carboxylated 1.3S due to exchange between two or more conformations within the N-terminus causing line broadening. It is proposed that interactions between the biotin-CO2-and the N-terminal amino acids perturb this conformational equilibrium causing some N-terminal residues to appear in the 1D NMR spectrum of the carboxylated form. Further details are apparent from a comparison of the 2D spectra of the 1.3S−CO2-and 1.3S proteins, where carboxylation causes several peaks from the C-terminal half to shift as well as the appearance of resonances due to some residues located at the N-terminal half of the protein. FTIR difference spectra were used also to follow spontaneous decarboxylation of the 1.3S−CO2-. For the carboxylated 1.3S, the difference spectra provided the vibrational signature of the CO2-on the biotin ring. A doublet was identified at 1695 and 1699 cm-1that increased in intensity with increasingt. This is assigned to an antisymmetric stretching vibration of the CO2-group bound to biotin on the 1.3S protein. Its position and profile provide further evidence for interactions occurring between the biotin-CO2-group and the 1.3S protein. These studies demonstrate the highly mobile, “poised” nature of the 1.3S protein engineered for its role as a CO2-translocator.