Department of Biochemistry, Molecular Biology, and Biophysics and The Biotechnology Institute, University of Minnesota, 1479 Gortner Avenue, St. Paul, Minnesota 55108, USA.
Biochemistry. 2010 Mar 9;49(9):1931-42. doi: 10.1021/bi9021268.
Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of epsilon-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher k(cat), but K(m) also increased so the specificity constant, k(cat)/K(m), remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of epsilon-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for larger alcohol moieties but binds epsilon-caprolactone more tightly. These results are consistent with the natural function of perhydrolases being either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones.
许多丝氨酸水解酶催化过水解反应，即羧酸和过氧化氢可逆地形成过氧酸。最近，我们发现，荧光假单胞菌（SIK WI）芳基酯酶（PFE）中醇结合口袋中的单个氨基酸取代，L29P，将 PFE 对过乙酸形成的特异性常数提高了>100 倍[Bernhardt 等人，（2005 年）Angew. Chem.，Int. Ed. 44，2742]。在本文中，我们扩展了这项工作，以解决以下三个问题。首先，过水解活性增加的分子基础是什么？我们之前提出，L29P 取代在过渡态中在酶和过氧化氢之间形成氢键。在这里，我们报告了 L29P PFE 的两个 X 射线结构，支持了这一假设。两个结构都显示出主链羰基氧更接近预期的活性部位丝氨酸。一个结构进一步显示，在酰基-酶机制的反应中，活性部位存在乙酸盐。我们还通过质谱检测到ε-己内酯水解的酰基-酶中间产物。其次，我们能否进一步提高过水解活性？我们发现，过氧乙酸的逆反应，即水解为乙酸和过氧化氢，几乎以扩散限制的速率发生。由于逆反应不能进一步增加，正反应也不能增加。与这一预测一致，两个具有额外氨基酸取代的变体显示出 2 倍更高的 k(cat)，但 K(m)也增加，因此特异性常数 k(cat)/K(m)仍然相似。第三，L29P 取代如何改变酯酶活性？大多数酯（乙酸乙酯 75 倍）的水解活性降低，但甲酯除外。相比之下，L29P PFE 催化ε-己内酯的水解效率比野生型 PFE 高五倍。分子建模表明，将羰基基团移近活性部位会阻止较大的醇部分进入，但会更紧密地结合ε-己内酯。这些结果与过水解酶的天然功能是过氧羧酸的水解或内酯的水解一致。
