Coolbear T, Whittaker J M, Daniel R M
Thermophile Research Unit, University of Waikato, Hamilton, New Zealand.
Biochem J. 1992 Oct 15;287 ( Pt 2)(Pt 2):367-74. doi: 10.1042/bj2870367.
The proteinase from the extremely thermophilic Bacillus strain EA.1 exhibits maximum stability at a pH of approx. 6.5. In the presence of calcium ions the half-life at 95 degrees C of the enzyme at this pH was 17 min, and loss of activity followed first-order decay kinetics. The role of metal ions in the activity and stability of the enzyme was studied using the holoenzyme, the metal-depleted apoenzyme, and a zinc-enriched apoenzyme preparation. Zinc and calcium ions were the preferred bivalent cations for the active site and stabilization site(s) respectively. Stabilization by metal ions was not in itself a highly stringent process, but ions other than calcium which stabilized the enzyme generally had a concomitant inhibitory effect on activity. Inhibition and stabilization of the enzyme by cations were concentration-dependent effects and certain ions activated the apoenzyme but not the holoenzyme. Manganese(II) ions conferred some stability and also activated the enzyme, but in the latter case were not as effective as zinc ions. The results are discussed with reference to the ionic radii, co-ordination number and preferred ligand donors of the ions. Mercury(II) ions severely compromised enzyme activity and stability, and the effects of thiol-reactive agents suggest that thiol groups also have a role in enzyme integrity.
来自极端嗜热芽孢杆菌菌株EA.1的蛋白酶在pH约为6.5时表现出最大稳定性。在钙离子存在下，该酶在此pH值下于95℃的半衰期为17分钟，活性丧失遵循一级衰变动力学。使用全酶、金属耗尽的脱辅基酶和富含锌的脱辅基酶制剂研究了金属离子在该酶活性和稳定性中的作用。锌离子和钙离子分别是活性位点和稳定位点的首选二价阳离子。金属离子的稳定作用本身并不是一个非常严格的过程，但除了钙离子外，能稳定该酶的离子通常对活性有伴随的抑制作用。阳离子对该酶的抑制和稳定作用是浓度依赖性效应，某些离子能激活脱辅基酶但不能激活全酶。锰（II）离子赋予了一定的稳定性并激活了该酶，但在后一种情况下不如锌离子有效。结合离子的离子半径、配位数和首选配体供体对结果进行了讨论。汞（II）离子严重损害了酶的活性和稳定性，硫醇反应剂的作用表明硫醇基团在酶的完整性中也起作用。
