Lamba Vandana, Sanchez Enis, Fanning Lauren Rose, Howe Kathryn, Alvarez Maria Alejandra, Herschlag Daniel, Forconi Marcello
Department of Biochemistry, Stanford University , Stanford, California 94305, United States.
Department of Chemistry and Biochemistry, College of Charleston , Charleston, South Carolina 29424, United States.
Biochemistry. 2017 Jan 31;56(4):582-591. doi: 10.1021/acs.biochem.6b00762. Epub 2017 Jan 20.
Kemp eliminases represent the most successful class of computationally designed enzymes, with rate accelerations of up to 10-fold relative to the rate of the same reaction in aqueous solution. Nevertheless, several other systems such as micelles, catalytic antibodies, and cavitands are known to accelerate the Kemp elimination by several orders of magnitude. We found that the naturally occurring enzyme ketosteroid isomerase (KSI) also catalyzes the Kemp elimination. Surprisingly, mutations of D38, the residue that acts as a general base for its natural substrate, produced variants that catalyze the Kemp elimination up to 7000-fold better than wild-type KSI does, and some of these variants accelerate the Kemp elimination more than the computationally designed Kemp eliminases. Analysis of the D38N general base KSI variant suggests that a different active site carboxylate residue, D99, performs the proton abstraction. Docking simulations and analysis of inhibition by active site binders suggest that the Kemp elimination takes place in the active site of KSI and that KSI uses the same catalytic strategies of the computationally designed enzymes. In agreement with prior observations, our results strengthen the conclusion that significant rate accelerations of the Kemp elimination can be achieved with very few, nonspecific interactions with the substrate if a suitable catalytic base is present in a hydrophobic environment. Computational design can fulfill these requirements, and the design of more complex and precise environments represents the next level of challenges for protein design.
肯普消除酶是计算设计酶中最成功的一类，相对于在水溶液中相同反应的速率，其反应速率加速可达10倍。然而，已知其他一些体系，如胶束、催化抗体和穴状配体，能将肯普消除反应加速几个数量级。我们发现天然存在的酶酮甾体异构酶（KSI）也能催化肯普消除反应。令人惊讶的是，作为其天然底物的通用碱的D38残基发生突变后，产生的变体催化肯普消除反应的效果比野生型KSI好7000倍，其中一些变体加速肯普消除反应的能力超过了计算设计的肯普消除酶。对D38N通用碱KSI变体的分析表明，另一个活性位点羧酸盐残基D99进行质子提取。对接模拟和活性位点结合剂抑制分析表明，肯普消除反应发生在KSI的活性位点，并且KSI使用与计算设计酶相同的催化策略。与先前的观察结果一致，我们的结果强化了这样一个结论：如果在疏水环境中存在合适的催化碱，与底物进行极少的非特异性相互作用就能实现肯普消除反应显著的速率加速。计算设计可以满足这些要求，而设计更复杂、精确的环境是蛋白质设计的下一个挑战层次。
