Understanding enzyme mechanisms is of paramount importance from both the basic biophysics perspective of understanding life processes and the role of enzymes in diseases. To achieve a detailed understanding of enzyme catalysis, the effects of protein structure and dynamics on the reaction energetics need to be elucidated. We propose a combined computational and experimental approach that combines the synthetic, computational and structural biology expertise of a team of investigators that has been working together for >15 years to create a ?molecular movie? where the position, movement and energy of every atom in the system followed over the entire reaction pathway. The proposal exploits the emerging convergence of timescales accessible by molecular simulation using GPUs and time resolved structural biology.
Specific Aim 1 describes the simulation of the complete reaction pathway of Pseudomonas mevalonii (Pm) HMGCoA Reductase (HMGR) and will use transition state force fields (TSFFs) generated by the quantum guided molecular mechanics method to allow the sec MD simulations of the chemical steps. TSFFs not only circumvent the well-known boundary problem of QM/MM, but are also 102-104 times faster. This allows a realistic modeling of the coupling of sec dynamics and catalysis that was demonstrated in the last grant period to be essential for understanding the reaction. Together with accelerated MD simulations of the conformational changes involved in the reaction using standard force fields, these computational studies cover the fsec to sec timescale.
In Specific Aim 2, the computational results will be merged with the results of a three-tiered approach to obtain structural snapshots with progressively increasing time resolution: (i) ?Frozen? intermediates that map out the overall pathway on long timescales, (ii) time resolved Laue crystallography using pH jump initiation on the msec timescale and (iii) use of photocaged substrates to allow time resolved Laue experiments on the sec timescale. This approach will be applied to the study of HMGR, an enzyme of high biophysical and biomedical significance that has a complex reaction mechanism involving three chemical steps, six large-scale conformational changes and two cofactor exchange steps. The project is highly innovative because it (i) uses a combination of MD simulations using TSFFs and time resolved crystallography to span timescales of at least 12 orders of magnitude, (ii) iteratively couples the Markov State analysis of long timescale trajectories to the Singular Value Decomposition used to analyze time resolved crystallography data, thus providing new tools to generate and experimentally validate trial structures (iii) applies global optimization and machine learning techniques to allow the automated fitting of TSFFs for proteins, which will enhance the application of this powerful method to other proteins and (iv) provides new photocaged substrates for the study of enzyme mechanisms to the chemical biology community. All tool compounds, methods and codes developed in this project will be made available to the scientific community.

Public Health Relevance

The detailed study of enzyme mechanisms is a cornerstone of biophysical chemistry that, while basic in nature, has had a major impact on human health including the development of new mechanism-based drugs for a range of diseases and an understanding of the mechanism of action for existing drugs that allows the design of combination therapies. The combination of Laue crystallography and long-scale MD simulations will allow simultaneous studies of structure, dynamics and energetic studies with unprecedented detail. The application to HMG CoA Reductase, arguably the single most important drug target in western industrialized countries, will demonstrate the applicability of the methodology to an enzyme of high mechanistic complexity. 1

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Macromolecular Structure and Function D Study Section (MSFD)
Program Officer
Lyster, Peter
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Notre Dame
Schools of Arts and Sciences
Notre Dame
United States
Zip Code
Rosales, Anthony R; Quinn, Taylor R; Wahlers, Jessica et al. (2018) Application of Q2MM to predictions in stereoselective synthesis. Chem Commun (Camb) 54:8294-8311
Hansen, Eric; Limé, Elaine; Norrby, Per-Ola et al. (2016) Anomeric Effects in Sulfamides. J Phys Chem A 120:3677-82
Hansen, Eric; Rosales, Anthony R; Tutkowski, Brandon et al. (2016) Prediction of Stereochemistry using Q2MM. Acc Chem Res 49:996-1005