Loop Motion in Triosephosphate Isomerase is not a Simple Open and Shut Case

Conformational changes are crucial for the catalytic action of many enzymes. A prototypical and well-studied example is loop opening and closure in triosephosphate isomerase (TIM), which is thought to determine the rate of catalytic turnover in many circumstances. Specifically, TIM loop 6 ‘grips’ the phosphodianion of the substrate and, together with a change in loop 7, sets up the TIM active site for efficient catalysis. Crystal structures of TIM typically show an open or a closed conformation of loop 6, with the tip of the loop moving ~7 Å between conformations. Many studies have interpreted this motion as a two-state, rigid-body transition. Here, we use extensive molecular dynamics simulations, with both conventional and enhanced sampling techniques, to analyze loop motion in apo and substrate-bound TIM in detail, using five crystal structures of the dimeric TIM from S. cerevisiae. We find that loop 6 is highly flexible and samples multiple conformational states. Empirical valence bond simulations of the first reaction step show that slight displacements away from the fully closed loop conformation can be sufficient to abolish much of the catalytic action; full closure is required for efficient reaction. The conformational change of the loops in TIM is thus not a simple ‘open and shut’ case, and is crucial for its catalytic action. Our detailed analysis of loop motion in a highly efficient enzyme highlights the complexity of loop conformational changes and their role in biological catalysis

A Multiscale Simulation Approach to Modeling Drug–Protein Binding Kinetics

Drug-target binding kinetics has recently emerged as a sometimes critical determinant of in vivo efficacy and toxicity. Its rational optimization to improve potency or reduce side effects of drugs is, however, extremely difficult. Molecular simulations can play a crucial role in identifying features and properties of small ligands and their protein targets affecting the binding kinetics, but significant challenges include the long time scales involved in (un)binding events and the limited accuracy of empirical atomistic force fields (lacking, e.g., changes in electronic polarization). In an effort to overcome these hurdles, we propose a method that combines state-of-the-art enhanced sampling simulations and quantum mechanics/molecular mechanics (QM/MM) calculations at the BLYP/VDZ level to compute association free energy profiles and characterize the binding kinetics in terms of structure and dynamics of the transition state ensemble. We test our combined approach on the binding of the anticancer drug Imatinib to Src kinase, a well-characterized target for cancer therapy with a complex binding mechanism involving significant conformational changes. The results indicate significant changes in polarization along the binding pathways, which affect the predicted binding kinetics. This is likely to be of widespread importance in binding of ligands to protein targets

The quest for accurate theoretical models of metalloenzymes: An aid to experiment

Enzymes are versatile oxidants in Nature that catalyze a range of reactions very efficiently. Experimental studies on the mechanism of enzymes are sometimes difficult due to the short lifetime of catalytic cycle intermediates. Theoretical modeling can assist and guide experiment and elucidate mechanisms for fast reaction pathways. Two key computational approaches are in the literature, namely quantum mechanics/molecular mechanics (QM/MM) on complete enzyme structures and QM cluster models on active site structures only. These two approaches are reviewed here. We give examples where the QM cluster approach worked well and, for instance, enabled the bioengineering of an enzyme to change its functionality. In addition, several examples are given, where QM cluster models were insufficient and full QM/MM structures were needed to establish regio-, chemo-, and stereoselectivities