Merkler D J, Srikumar K, Marchese-Ragona S P, Wedler F C
Department of Molecular and Cell Biology, P.M. Althouse Laboratory, Pennsylvania State University, University Park 16802.
Biochim Biophys Acta. 1988 Jan 4;952(1):101-14. doi: 10.1016/0167-4838(88)90106-9.
The extreme thermophile, Bacillus caldolyticus, contains two regulatory isoforms of glutamine synthetase (glutamate-ammonia ligase, EC 6.3.1.2), E-I and E-II, produced as separate gene products. Light scattering and electron microscopy data indicate that these thermophilic enzymes aggregate to higher molecular weight species in two stages: initial polymerization of native dodecamers, followed by 'melting' of the aggregated species to produce amorphous denatured protein. The initial stages of the aggregation occurred at temperatures below those for time-dependent denaturation, especially for E-II. In contrast, mesophilic (B. subtilis) enzyme showed no evidence of temperature-dependent aggregation. Thus, aggregation may be a stabilizing mechanism for the thermophilic systems. Bound metal ions and substrates caused dramatic increases in the temperatures at which aggregation and loss of activity occurred for thermophilic enzymes. Certain combinations of ligands (e.g., MnATP + L-glutamate) acted synergistically, so that these complexes denatured only above 90 degrees C. Various models were considered for heat-driven aggregation followed by denaturation, plus ligand stabilization. Taken together, the data are most consistent with unfolding of subunits within the dodecameric unit, rather than unfolding to monomers prior to aggregation.
嗜热芽孢杆菌（Bacillus caldolyticus）含有谷氨酰胺合成酶（谷氨酸-氨连接酶，EC 6.3.1.2）的两种调节同工型E-I和E-II，它们是作为单独的基因产物产生的。光散射和电子显微镜数据表明，这些嗜热酶分两个阶段聚集成更高分子量的物种：天然十二聚体的初始聚合，随后聚集物种的“熔化”以产生无定形变性蛋白。聚集的初始阶段发生在低于时间依赖性变性的温度下，特别是对于E-II。相比之下，嗜温菌（枯草芽孢杆菌）的酶没有显示出温度依赖性聚集的证据。因此，聚集可能是嗜热系统的一种稳定机制。结合的金属离子和底物导致嗜热酶发生聚集和活性丧失的温度显著升高。某些配体组合（例如，MnATP + L-谷氨酸）具有协同作用，因此这些复合物仅在90℃以上变性。考虑了各种热驱动聚集后变性以及配体稳定化的模型。综合来看，数据最符合十二聚体单元内亚基的解折叠，而不是在聚集之前解折叠为单体。
