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首页> 外文期刊>Chemistry: A European journal >Revised molecular basis of the promiscuous carboxylic acid perhydrolase activity in serine hydrolases
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Revised molecular basis of the promiscuous carboxylic acid perhydrolase activity in serine hydrolases

机译：修订的丝氨酸水解酶中混杂的羧酸过水解酶活性的分子基础

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Several serine hydrolases catalyze a promiscuous reaction: perhydrolysis of carboxylic acids to form peroxycarboxylic acids. The working hypothesis is that perhydrolases are more selective than esterases for hydrogen peroxide over water. In this study, we tested this hypothesis, and focused on L29P-PFE (Pseudomonas fluorescens esterase), which catalyzes perhydrolysis of acetic acid 43-fold faster than wild-type PFE. This hypothesis predicts that L29P-PFE should be approximately 43-fold more selective for hydrogen peroxide than wild-type PFE, but experiments show that L29P-PFE is less selective. The ratio of hydrolysis to perhydrolysis of methyl acetate at different concentrations of hydrogen peroxide fit a kinetic model for nucleophile selectivity. L29P-PFE (β _0=170 M ~(-1)) is approximately half as selective for hydrogen peroxide over water than wild-type PFE (β _0=330 M ~(-1)), which contradicts the working hypothesis. An alternative hypothesis is that carboxylic acid perhydrolases increase perhydrolysis by forming the acyl-enzyme intermediate faster. Consistent with this hypothesis, the rate of acetyl-enzyme formation, measured by ~(18)O-water exchange into acetic acid, was 25-fold faster with L29P-PFE than with wild-type PFE, which is similar to the 43-fold faster perhydrolysis with L29P-PFE. Molecular modeling of the first tetrahedral intermediate (T _d1) suggests that a closer carbonyl group found in perhydrolases accepts a hydrogen bond from the leaving group water. This revised understanding can help design more efficient enzymes for perhydrolysis and shows how subtle changes can create new, unnatural functions in enzymes.
机译：几种丝氨酸水解酶催化混合反应：羧酸的过水解形成过氧羧酸。可行的假设是过水解酶比过氧化氢对酯酶的选择性更高。在这项研究中，我们检验了这一假设，并侧重于L29P-PFE（荧光假单胞菌酯酶），该酶催化乙酸的过水解比野生型PFE快43倍。该假设预测L29P-PFE对过氧化氢的选择性应比野生型PFE高约43倍，但实验表明L29P-PFE的选择性较差。在不同浓度的过氧化氢下，乙酸甲酯的水解与全水解之比符合亲核试剂选择性的动力学模型。 L29P-PFE（β_0 = 170 M〜（-1））对水的过氧化氢选择性约为野生型PFE（β_0 = 330 M〜（-1）），这与工作假设相矛盾。另一种假设是羧酸过水解酶通过更快地形成酰基酶中间体来增加过水解。与此假设相符，L29P-PFE的〜（18）O-水交换成乙酸后测得的乙酰酶形成速率比野生型PFE快25倍，与43-用L29P-PFE可以更快地进行过水解。第一个四面体中间体的分子模型（T _d1）表明，在全水解酶中发现的更接近的羰基接受离去基团水的氢键。修订后的理解可以帮助设计用于更高效水解的酶，并显示出细微的变化如何在酶中产生新的非自然功能。

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