Daniel R M, Dines M, Petach H H
Department of Biological Sciences, University of Walkato, Hamilton, New Zealand.
Biochem J. 1996 Jul 1;317 ( Pt 1)(Pt 1):1-11. doi: 10.1042/bj3170001.
Now that enzymes are available that are stable above 100 degrees C it is possible to investigate conformational stability at this temperature, and also the effect of high-temperature degradative reactions in functioning enzymes and the inter-relationship between degradation and denaturation. The conformational stability of proteins depends upon stabilizing forces arising from a large number of weak interactions, which are opposed by an almost equally large destabilizing force due mostly to conformational entropy. The difference between these, the net free energy of stabilization, is relatively small, equivalent to a few interactions. The enhanced stability of very stable proteins can be achieved by an additional stabilizing force which is again equivalent to only a few stabilizing interactions. There is currently no strong evidence that any particular interaction (e.g. hydrogen bonds, hydrophobic interactions) plays a more important role in proteins that are stable at 100 degrees C than in those stable at 50 degrees C, or that the structures of very stable proteins are systematically different from those of less stable proteins. The major degradative mechanisms are deamidation of asparagine and glutamine, and succinamide formation at aspartate and glutamate leading to peptide bond hydrolysis. In addition to being temperature-dependent, these reactions are strongly dependent upon the conformational freedom of the susceptible amino acid residues. Evidence is accumulating which suggests that even at 100 degrees C deamidation and succinamide formation proceed slowly or not at all in conformationally intact (native) enzymes. Whether this is the case at higher temperatures is not yet clear, so it is not known whether denaturation of degradation will set the upper limit of stability for enzymes.
既然有了在100摄氏度以上仍保持稳定的酶，就有可能研究该温度下的构象稳定性，以及高温降解反应对功能性酶的影响，还有降解与变性之间的相互关系。蛋白质的构象稳定性取决于大量弱相互作用产生的稳定力，而与之抗衡的是几乎同样大的去稳定力，主要源于构象熵。二者之差，即稳定化净自由能，相对较小，相当于少数几个相互作用。非常稳定的蛋白质所增强的稳定性可通过一种额外的稳定力来实现，这种稳定力同样也只相当于少数几个稳定相互作用。目前尚无有力证据表明，在100摄氏度稳定的蛋白质中，任何特定相互作用（如氢键、疏水相互作用）比在50摄氏度稳定的蛋白质中发挥更重要的作用，或者非常稳定的蛋白质结构与稳定性较差的蛋白质结构有系统性差异。主要的降解机制是天冬酰胺和谷氨酰胺的脱酰胺作用，以及天冬氨酸和谷氨酸处形成琥珀酰胺导致肽键水解。除了依赖温度外，这些反应还强烈依赖于敏感氨基酸残基的构象自由度。越来越多的证据表明，即使在100摄氏度，脱酰胺作用和琥珀酰胺形成在构象完整（天然）的酶中也进行得很慢或根本不发生。在更高温度下是否如此尚不清楚，因此也不知道降解导致的变性是否会设定酶稳定性的上限。
