Transition state stabilization and substrate strain in enzyme catalysis: ab initio QM/MM modelling of the chorismate mutase reaction

Abstract

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.

QM/MM modelling of oleamide hydrolysis in fatty acid amide hydrolase (FAAH) reveals a new mechanism of nucleophile activation

“…The geometry corresponding to configuration 2 is not a stationary point, indicating that the two proton transfers, leading to the nucleophilic Ser241 alcoholate ion, are concerted. The calculated barrier at the higher level (18 kcal mol 21 ) is close to the experimentally deduced activation barrier of y16 kcal mol 21 . 10 The structure of the transition state (the highest energy point along the minimum energy path), and of neighbouring species, shows a proton being transferred from Ser241 to Ser217, i.e.…”

Comparison of DFT and ab initio QM/MM methods for modelling reaction in chorismate synthase

“…FMN was modified to FMNH − and all atom types in the topology files were assigned based on the CHARMM27 parameter set [18]. The MM atomic partial charges of FMNH − and EPSP used in the initial setup of the model were based on fitting point charges to the electrostatic potential (HF/6-31G(d) CHELPG) [19,20].There are four different forms of the phosphate anion: PO 4 3− , HPO 4 2− , H 2 PO 4 − , and H 3 PO 4. Under neutral conditions, the phosphate anion could exist in either the H 2 PO 4 − or the HPO 4 2− forms, so the protonation state of the phosphate group of EPSP is an important consideration for the setup of the model system.…”

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

“…A second type of substrate-assisted catalysis may result from internal mobility of the paminobenzoylglutamate moiety of the DHF. By allowing movement of the large benzoyl group while constraining the position of the pteridine ring, the C6 position of the pteridine could be subject to forces which may strain the ring structure and move the system toward a more reactive transition state structure (57,58). In this view of catalysis, dynamics of the bound substrate complex could ultimately lead to transition state formation (59, 60).…”