Ranaghan Kara E, Ridder Lars, Szefczyk Borys, Sokalski W Andrzej, Hermann Johannes C, Mulholland Adrian J
Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol, UK BS8 1TS.
Org Biomol Chem. 2004 Apr 7;2(7):968-80. doi: 10.1039/b313759g. Epub 2004 Mar 3.
To investigate fundamental features of enzyme catalysis, there is a need for high-level calculations capable of modelling crucial, unstable species such as transition states as they are formed within enzymes. We have modelled an important model enzyme reaction, the Claisen rearrangement of chorismate to prephenate in chorismate mutase, by combined ab initio quantum mechanics/molecular mechanics (QM/MM) methods. The best estimates of the potential energy barrier in the enzyme are 7.4-11.0 kcal mol(-1)(MP2/6-31+G(d)//6-31G(d)/CHARMM22) and 12.7-16.1 kcal mol(-1)(B3LYP/6-311+G(2d,p)//6-31G(d)/CHARMM22), comparable to the experimental estimate of Delta H(++)= 12.7 +/- 0.4 kcal mol(-1). The results provide unequivocal evidence of transition state (TS) stabilization by the enzyme, with contributions from residues Arg90, Arg7, and Arg63. Glu78 stabilizes the prephenate product (relative to substrate), and can also stabilize the TS. Examination of the same pathway in solution (with a variety of continuum models), at the same ab initio levels, allows comparison of the catalyzed and uncatalyzed reactions. Calculated barriers in solution are 28.0 kcal mol(-1)(MP2/6-31+G(d)/PCM) and 24.6 kcal mol(-1)(B3LYP/6-311+G(2d,p)/PCM), comparable to the experimental finding of Delta G(++)= 25.4 kcal mol(-1) and consistent with the experimentally-deduced 10(6)-fold rate acceleration by the enzyme. The substrate is found to be significantly distorted in the enzyme, adopting a structure closer to the transition state, although the degree of compression is less than predicted by lower-level calculations. This apparent substrate strain, or compression, is potentially also catalytically relevant. Solution calculations, however, suggest that the catalytic contribution of this compression may be relatively small. Consideration of the same reaction pathway in solution and in the enzyme, involving reaction from a 'near-attack conformer' of the substrate, indicates that adoption of this conformation is not in itself a major contribution to catalysis. Transition state stabilization (by electrostatic interactions, including hydrogen bonds) is found to be central to catalysis by the enzyme. Several hydrogen bonds are observed to shorten at the TS. The active site is clearly complementary to the transition state for the reaction, stabilizing it more than the substrate, so reducing the barrier to reaction.
为了研究酶催化的基本特征，需要进行高水平的计算，以便对关键的不稳定物种（如酶内形成的过渡态）进行建模。我们通过组合从头算量子力学/分子力学（QM/MM）方法，对一个重要的模型酶反应——分支酸变位酶中分支酸向预苯酸的克莱森重排反应进行了建模。酶中势能垒的最佳估计值为7.4 - 11.0千卡摩尔⁻¹（MP2/6 - 31 + G(d)//6 - 31G(d)/CHARMM22）和12.7 - 16.1千卡摩尔⁻¹（B3LYP/6 - 311 + G(2d,p)//6 - 31G(d)/CHARMM22），与实验估计的ΔH⁺⁺ = 12.7 ± 0.4千卡摩尔⁻¹相当。结果明确证明了酶对过渡态（TS）的稳定作用，其中Arg90、Arg7和Arg63残基起到了作用。Glu78稳定了预苯酸产物（相对于底物），也能稳定过渡态。在相同的从头算水平下，用各种连续介质模型对溶液中的相同反应途径进行研究，从而可以比较催化反应和非催化反应。溶液中计算得到的能垒为28.0千卡摩尔⁻¹（MP2/6 - 31 + G(d)/PCM）和24.6千卡摩尔⁻¹（B3LYP/6 - 311 + G(2d,p)/PCM），与实验发现的ΔG⁺⁺ = 25.4千卡摩尔⁻¹相当，并且与实验推导的酶使反应速率加速10⁶倍一致。发现底物在酶中发生了显著变形，采用了更接近过渡态的结构，尽管压缩程度小于低水平计算的预测值。这种明显的底物应变或压缩可能在催化中也具有相关性。然而，溶液计算表明这种压缩的催化贡献可能相对较小。对溶液和酶中相同反应途径的研究，涉及底物“近攻击构象”的反应，表明采用这种构象本身对催化的贡献不大。发现过渡态稳定化（通过包括氢键在内的静电相互作用）是酶催化的核心。在过渡态观察到几个氢键缩短。活性位点显然与反应的过渡态互补，对其稳定作用大于对底物的稳定作用，从而降低了反应的能垒。
