Giordano A, Cannio R, La Cara F, Bartolucci S, Rossi M, Raia C A
Institute of Protein Biochemistry and Enzymology, CNR, Naples, Italy.
Biochemistry. 1999 Mar 9;38(10):3043-54. doi: 10.1021/bi982326e.
A mutant of the thermostable NAD+-dependent homotetrameric alcohol dehydrogenase from Sulfolobus solfataricus (SsADH), which has a single substitution, Asn249Tyr, located at the coenzyme binding domain, was obtained by error prone PCR. The mutant enzyme, which was purified from Escherichia coli to homogeneous form, exhibits a specific activity that is more than 6-fold greater than that of the wild type enzyme, as measured at 65 degrees C with benzyl alcohol as the substrate. The oxidation rate of aliphatic alcohols and the reduction rate of aromatic aldehydes were also higher. The dissociation constants for NAD+ and NADH determined at 25 degrees C and pH 8.8 were 3 orders of magnitude greater compared to those of the wild type enzyme. It is thought that the higher turnover of the mutant SsADH is due to the faster dissociation of the modified enzyme-coenzyme complex. Spectroscopic studies showed no relevant changes in either secondary or tertiary structure, while analysis with fluorescent probes revealed a significant increase in surface hydrophobicity for the mutant, with respect to that of the wild type molecule. The mutant SsADH displays improved thermal stability, as indicated by the increase in Tm from 90 to 93 degrees C, which was determined by the apparent transition curves. Kinetic thermal stability studies at pH 9.0 for mutant SsADH showed a marked increase in activation enthalpy compensated by an entropy gain, which resulted in a higher activation barrier against thermal unfolding of the enzyme. Ammonia analysis showed that the Asn249Tyr substitution produced the effect of markedly reducing the extent of deamidation during thermoinactivation, thus suggesting that Asn249 plays a significant role in the mechanism of irreversible thermal denaturation of the archaeal ADH. Furthermore, the decrease in the activating effect by moderate concentrations of denaturants and studies with proteases and chelating agents point to an increase in structural rigidity and a tightening of structural zinc as additional factors responsible for the improved thermal resistance of the mutant enzyme.
通过易错PCR获得了来自嗜热栖热菌（SsADH）的耐热NAD⁺依赖性同四聚体醇脱氢酶的一个突变体，该突变体在辅酶结合结构域有一个单取代，即Asn249Tyr。从大肠杆菌中纯化至均一形式的突变酶，以苯甲醇为底物在65℃下测定时，其比活性比野生型酶高6倍以上。脂肪醇的氧化速率和芳香醛的还原速率也更高。在25℃和pH 8.8下测定的NAD⁺和NADH的解离常数比野生型酶大3个数量级。据认为，突变体SsADH的更高周转率是由于修饰的酶 - 辅酶复合物的更快解离。光谱研究表明二级或三级结构均无相关变化，而用荧光探针分析显示突变体相对于野生型分子表面疏水性显著增加。突变体SsADH表现出改善的热稳定性，由表观转变曲线测定的熔解温度（Tm）从90℃增加到93℃表明了这一点。在pH 9.0下对突变体SsADH进行的动力学热稳定性研究表明，活化焓显著增加，同时熵增加得到补偿，这导致了更高的抗酶热解折叠活化能垒。氨分析表明，Asn249Tyr取代产生了显著降低热失活过程中脱酰胺程度的效果，因此表明Asn249在古菌ADH不可逆热变性机制中起重要作用。此外，中等浓度变性剂活化作用的降低以及蛋白酶和螯合剂的研究表明，结构刚性增加和结构锌的收紧是导致突变酶耐热性提高的额外因素。
