Origins of catalysis by computationally designed retroaldolase enzymes
Origins of catalysis by computationally designed retroaldolase enzymes
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DOI：
10.1073/pnas.0913638107
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发表时间：
2010-03-16
影响因子：
11.1
通讯作者：
Herschlag, Daniel
中科院分区：
文献类型：
--
作者：
Lassila, Jonathan K.;Baker, David;Herschlag, Daniel
我们已经研究了最近报道的计算设计的恢复氧化酶酶，目的是了解其催化能力的程度和起源。在溶液中，设计酶与原代胺催化剂的直接比较表明，最活跃的恢复倍醛酶的速率加速度为10（5）。通过对设计的恢复醇酶的pH速率研究以及一系列胺催化剂的布朗斯特相关性的评估，我们发现酶的赖氨酸PK（a）值在酶中的3-4个单位转移了3-4个单位，但是从转移的PKA催化贡献值估计为适中，约10倍。对于最活跃的酶，我们评估了另外两个设计成分的催化贡献：旨在稳定结合水分子和疏水底物结合相互作用的基序。突变分析表明，绑定的水基序不导致速率加速度。比较设计的底物相对于最小底物的速率加速度的比较表明，疏水底物结合相互作用约为酶促加速度约10（3）倍。总之，这些结果表明底物结合相互作用并转移催化赖氨酸的PK（A）可以解释酶的大部分速率加速度。其他观察结果表明，这些相互作用在底物和活性位点催化基团的特异性方面受到限制。因此，未来的设计工作可能会受益于在结合相互作用和催化群体的放置方面的精确度中受益。
We have investigated recently reported computationally designed retroaldolase enzymes with the goal of understanding the extent and the origins of their catalytic power. Direct comparison of the designed enzymes to primary amine catalysts in solution revealed a rate acceleration of 10(5)-fold for the most active of the designed retroaldolases. Through pH-rate studies of the designed retroaldolases and evaluation of a Bronsted correlation for a series of amine catalysts, we found that lysine pK(a) values are shifted by 3-4 units in the enzymes but that the catalytic contributions from the shifted pKa values are estimated to be modest, about 10-fold. For the most active of the reported enzymes, we evaluated the catalytic contribution of two other design components: a motif intended to stabilize a bound water molecule and hydrophobic substrate binding interactions. Mutational analysis suggested that the bound water motif does not contribute to the rate acceleration. Comparison of the rate acceleration of the designed substrate relative to a minimal substrate suggested that hydrophobic substrate binding interactions contribute around 10(3)-fold to the enzymatic rate acceleration. Altogether, these results suggest that substrate binding interactions and shifting the pK(a) of the catalytic lysine can account for much of the enzyme's rate acceleration. Additional observations suggest that these interactions are limited in the specificity of placement of substrate and active site catalytic groups. Thus, future design efforts may benefit from a focus on achieving precision in binding interactions and placement of catalytic groups.