Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London, UK.
J Biotechnol. 2011 Sep 10;155(2):209-16. doi: 10.1016/j.jbiotec.2011.06.023. Epub 2011 Jun 23.
We have previously shown that the denaturation of TK with urea follows a non-aggregating though irreversible denaturation pathway in which the cofactor binding appears to become altered but without dissociating, then followed at higher urea by partial denaturation of the homodimer prior to any further unfolding or dissociation of the two monomers. Urea is not typically present during biocatalysis, whereas access to TK enzymes that retain activity at increased temperature and extreme pH would be useful for operation under conditions that increase substrate and product stability or solubility. To provide further insight into the underlying causes of its deactivation in process conditions, we have characterised the effects of temperature and pH on the structure, stability, aggregation and activity of Escherichia coli transketolase. The activity of TK was initially found to progressively improve after pre-incubation at increasing temperatures. Loss of activity at higher temperature and low pH resulted primarily from protein denaturation and subsequent irreversible aggregation. By contrast, high pH resulted in the formation of a native-like state that was only partially inactive. The apo-TK enzyme structure content also increased at pH 9 to converge on that of the holo-TK. While cofactor dissociation was previously proposed for high pH deactivation, the observed structural changes in apo-TK but not holo-TK indicate a more complex mechanism.
我们之前已经表明，TK 在脲中的变性遵循非聚集但不可逆的变性途径，其中辅因子结合似乎发生改变但没有解离，然后在较高的脲浓度下，同二聚体发生部分变性，然后再进一步展开或解离两个单体。脲通常不在生物催化过程中存在，而获得在高温和极端 pH 值下保持活性的 TK 酶对于在增加底物和产物稳定性或溶解度的条件下操作将是有用的。为了更深入地了解其在过程条件下失活的根本原因，我们已经研究了温度和 pH 值对大肠杆菌转酮醇酶的结构、稳定性、聚集和活性的影响。最初发现，TK 的活性在逐渐提高的温度下预孵育后逐渐提高。在较高温度和低 pH 值下失去活性主要是由于蛋白质变性和随后的不可逆聚集。相比之下，高 pH 值导致形成类似于天然状态的部分失活状态。apo-TK 酶结构含量也在 pH 值为 9 时增加，与 holo-TK 趋同。虽然先前提出了高 pH 值失活时辅因子解离，但在 apo-TK 中观察到的结构变化而不是在 holo-TK 中观察到的结构变化表明存在更复杂的机制。
