Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis.
Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis.
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Mg2 连接寡聚化调节耻垢分枝杆菌 Lon (La) 蛋白酶的催化活性。
DOI：
10.1021/bi0102508
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Shrader,TE
中科院分区：
文献类型：
--
作者：
Rudyak,SG;Brenowitz,M;Shrader,TE
Lon （La）蛋白酶是由ATP和Mg2+离子激活的多聚酶，并受到α-酪蛋白等未折叠蛋白的刺激。耻垢分枝杆菌的Lon蛋白酶（Ms-Lon）的肽酶活性取决于其浓度和Mg2+的浓度。α-酪蛋白的加入部分替代了Mg2+激活酶。在化学解离实验中，加入α-酪蛋白后，需要较高浓度的尿素才能抑制Ms-Lon的催化活性。利用分析性超离心直接探讨了肽酶活性激活剂对Ms-Lon自结合的影响。沉降速度实验表明，Ms-Lon单体与低聚形式的蛋白质处于可逆平衡状态，Mg2+离子促进了自结合反应，而AMP-PNP或atp - γ s则不促进自结合反应。100 mM NaCl有利于低聚，并在次优浓度的MgCl2下刺激肽酶活性。沉淀平衡分析表明，在pH 8.0和20°C条件下，Ms-Lon在50 mM Tris和10 mM MgCl2条件下与六聚体结合，且组装反应依赖于Mg2+；在10 mM EDTA中，六聚体的摩尔分数随着mgcl2的降低而降低到检测不到的水平。在一系列初始蛋白质和mgcl2浓度下进行的实验分析得出两种组装模型：二聚体↔四聚体↔六聚体和定时器↔六聚体，与数据同样一致。有限的胰蛋白酶消化、CD和色氨酸荧光表明，在Mg2+连接的寡聚化过程中，二级和三级结构只有轻微的变化。这些结果表明，Ms-Lon肽酶活性的激活需要寡聚化，而Ms-Lon自结合反应是由其激活剂Mg2+和刺激剂未折叠蛋白促进的。
Lon (La) proteases are multimeric enzymes that are activated by ATP and Mg2+ions and stimulated by unfolded proteins such as α-casein. The peptidase activity of the Lon protease fromMycobacterium smegmatis(Ms-Lon) is dependent upon both its concentration and that of Mg2+. Addition of α-casein partially substitutes for Mg2+in activating the enzyme. In chemical dissociation experiments, higher concentrations of urea were required to inhibit Ms-Lon's catalytic activities after an addition of α-casein. Analytical ultracentrifugation was used to directly probe the effect of activators of peptidase activity on Ms-Lon self-association. Sedimentation velocity experiments reveal that Ms-Lon monomers are in a reversible equilibrium with oligomeric forms of the protein and that the self-association reaction is facilitated by Mg2+ions but not by AMP-PNP or ATPγS. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrations of MgCl2. Sedimentation equilibrium analysis shows that Ms-Lon associates to a hexamer at 50 mM Tris and 10 mM MgCl2, at pH 8.0 and 20 °C, and that the assembly reaction is Mg2+dependent; the mole fraction of hexamer decreases with decreasing MgCl2to undetectable levels in 10 mM EDTA. The analysis of experiments conducted at a series of initial protein and MgCl2concentrations yields two assembly models: dimer ↔ tetramer ↔ hexamer and timer ↔ hexamer, equally consistent with the data. Limited trypsin digestion, CD, and tryptophan fluorescence suggest only minor changes in secondary and tertiary structure upon Mg2+-linked oligomerization. These results show that activation of Ms-Lon peptidase activity requires oligomerization and that Ms-Lon self-association reaction is facilitated by its activator, Mg2+, and stimulator, unfolded protein.