Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with oxygen: chemistry and regulation by ligands.
Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with oxygen: chemistry and regulation by ligands.
复制标题
枯草芽孢杆菌谷氨酰胺磷酸核糖焦磷酸酰胺转移酶与氧的反应：配体的化学和调节。
DOI：
10.1021/bi00523a006
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发表时间：
1981
期刊：
影响因子：
2.9
通讯作者：
Switzer,RL
中科院分区：
文献类型：
--
作者：
Bernlohr,DA;Switzer,RL
摘要：枯草芽孢杆菌生长稳定期谷氨酸磷酸核糖焦磷酸氨基转移酶的铁硫中心与O2反应失活是该酶生理调节的重要方式。对纯化的酶在02氧化过程中的化学和物理变化进行了研究。4Fe-4S中心的铁被氧化成酶结合的高自旋Fe3+；S2“被氧化成硫代半胱氨酸和未知产物的混合物。氧化剂似乎是02，而不是过氧化氢、超氧化物、羟基自由基或单线态氧。氧化酶的大体物理变化表现为其聚集、溶解度降低和圆二色谱改变。描述了影响氧化活化速率的实验变量，其中最重要的是变构抑制剂AMP、ADP、GMP和GDP以及底物P-Rib-PP对失活速率的调节。AMP是一种有效的稳定剂，其作用可被P-Rib-PP拮抗。其他核苷酸，无论是单独作用还是协同作用，都是不稳定剂，并能增强AMP的稳定作用。结果从调节氨基转移酶的稳定性及其在体内的降解方面进行了讨论。E嘌呤生物合成的第一个酶，谷氨酰胺磷酸磷酸焦磷酸氨基转移酶(EC 2.4。2.14)在枯草芽孢杆菌中不仅受到抑制(Nishikawa等人，1967)和最终产物的抑制(Meyer&Switzer，1979)，而且还受到生长静止阶段细胞中氧依赖的失活(Turnbough&Switzer，1975a)的调节。氧依赖失活的本质是通过在无细胞提取物中与氧反应使氨基转移酶失活(Turnbough&Switzer，1975b)，以及随后证明纯酰胺转移酶是一种不稳定氧的铁硫蛋白(Wong等人，1977；Averill等人，1980)。氨基转移酶的失活是氧与铁硫中心反应的结果。伴随着失活的是铁硫发色团的漂白(Wong等人，1977)，无机硫化物的损失(Switzer等人，1979a，b)，以及铁从分析为Fe2+的形式转化为Fe3+(Switzer等人，1979a，b)。这个过程不需要“失活酶”。观察到氨基转移酶的失活速率受到变构配体的强烈影响，这为氨基转移酶失活的调控提供了线索(Turnbough&Switzer，1975b)。本文全面研究了高纯度氨基转移酶氧依赖失活所引起的化学和物理变化，以及底物、变构配体和其他潜在调节剂对酶活性的调节。根据实验结果，提出了氨基转移酶在体内对02稳定性的调控模型。
David A. Bernlohr and Robert L. Switzer* abstract: The inactivationof glutaminephosphoribosyl-pyrophosphate amidotransferase by reaction of its iron-sulfur center with 02 is believed to be a physiologically important mode of regulation of this enzyme in Bacillus subtilis cells in the stationary phase of growth. Chemical and physical changes accompanyingoxidation of the purified enzyme by 02 were studied. The iron of the 4Fe-4S center was oxidized to enzyme-bound high-spin Fe3+; the S2" was oxidized to a mixture of Sbound as thiocystine and unidentified products. The oxidant appeared to be 02, rather than peroxide, super-oxide, hydroxyl radical, or singlet oxygen. Gross physical changes in the oxidized enzyme were shown by its aggregation, decreased solubility, and altered circular dichroicspectrum. Experimental variables affecting the rate of oxidative inac-tivation were described; the most important of these was modulation of rates of inactivation by the allosteric inhibitors AMP, ADP, GMP, and GDP and by the substrate P-Rib-PP. AMP was a potent stabilizer, whose effect was antagonized by P-Rib-PP. The other nucleotides, either acting singly or acting as synergistic pairs, were destabilizers and able to an-tagonize stabilization by AMP. The results are discussed in terms of the regulation of the stability of amidotransferaseand its degradation in vivo. e first enzyme of purine biosynthesis, glutamine phos-phoribosylpyrophosphate amidotransferase (EC 2.4. 2.14), hereafter called “amidotransferase”, is regulated in Bacillus subtilis not only by repression (Nishikawa et al., 1967) and end-product inhibition (Meyer & Switzer, 1979) but also by oxygen-dependent inactivation in cells in the stationary phase of growth (Turnbough & Switzer, 1975a). The nature of the oxygen-dependent inactivation was clarified by the discovery that amidotransferase is inactivated by reaction with oxygen in cell-free extracts (Turnbough & Switzer, 1975b) and the subsequent demonstration that pure amidotransferase is an oxygen-labile iron-sulfur protein (Wong et al., 1977; Averill et al., 1980). Inactivation of amidotransferaseresults from reaction of oxygen with the iron-sulfur center. Inactivation is accompanied by bleaching of the iron-sulfur chromophore (Wong et al., 1977), loss of inorganic sulfide (Switzer et al., 1979a, b), and conversion of the iron from a form assaying as Fe2+ to Fe3+(Switzer et al., 1979a, b). No “inactivating enzyme” is required for this process. A clue to the regulation of the inactivationof amidotransferase was provided by the observation that the rate of inactivation of the enzyme in crude extracts was strongly affected by allosteric ligands (Turnbough & Switzer, 1975b). This paper presents the results of a complete study of the chemical and physical changes accom-panying oxygen-dependent inactivationof highly purified amidotransferase and of the modulation of the rate of inac-tivation by substrates, allosteric ligands, and other potential regulators. A tentative model for the regulation of the stability of amidotransferase to 02 in vivo has been deduced from the results.