Sun M M, Tolliday N, Vetriani C, Robb F T, Clark D S
Department of Chemical Engineering, University of California, Berkeley 94720, USA.
Protein Sci. 1999 May;8(5):1056-63. doi: 10.1110/ps.8.5.1056.
In this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophile Pyrococcus furiosus (Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C-terminus (rGDHt). The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance (ESR) spectroscopy of a spin-label attached to the terminal cysteine of rGDHt revealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36-fold for rGDHt at 105 degrees C and 750 atm, the largest pressure-induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDHt was also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure-induced thermostabilization is proposed.
在本文中，发现高达750个大气压（1个大气压 = 101千帕）的高压对两种极端嗜热谷氨酸脱氢酶（GDHs）具有很强的稳定作用：来自嗜热栖热菌（Pf）的天然酶，以及一种在C端含有额外四肽的重组GDH突变体（rGDHt）。四肽的存在在常压下极大地使重组突变体不稳定；然而，施加压力后，这种不稳定作用在很大程度上得到了逆转。对连接到rGDHt末端半胱氨酸的自旋标记进行电子自旋共振（ESR）光谱分析，结果显示其具有高度的流动性，这表明不稳定是由于四肽的热波动引起的亚基间离子对相互作用减弱所致。对于这两种酶，压力的稳定作用随温度和压力的升高而增强，在105摄氏度和750个大气压下，rGDHt的稳定作用达到36倍，这是迄今为止报道的最大的压力诱导酶热稳定性。通过添加甘油也实现了天然GDH和rGDHt的稳定。基于热失活动力学以及甘油对蛋白质结构的已知影响，提出了压力诱导热稳定性的机制。
