Hybrid Quantum Mechanical and Molecular Mechanical Studies of the Reaction Mechanism of Lactate and Malate Dehydrogenase


Principal Investigator

Jill Gready

Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research

Enzymes are proteins which are responsible for virtually all chemical reactions in cells. They bind
reactants (substrates) with a high degree of specificity, and chemically transform them with a phenomenal rate acceleration compared to the analogous reaction in solution. It is of fundamental interest to characterize the unique structural and energetic properties of the enzyme active site which enable this binding and catalysis. This project focuses on the reaction mechanism of two important glycolytic enzymes, lactate dehydrogenase (LDH) and malate dehydrogenase (MDH). LDH converts pyruvate to L-lactate in the presence of the cofactor NADH; similarly, MDH converts oxaloacetate to L-malate.
The relatively new hybrid quantum mechanical and molecular mechanical (QM/MM) technique is employed in this study. The computational cost of modelling a chemical reaction in an enzyme is minimized by partitioning the system: the active site and the substrate are treated quantum mechanically, while the protein environment is simulated using classical molecular mechanics. The two regions are coupled via approximate, parameterized interactions. The QM/MM approach thus takes advantage of the chemical accuracy of quantum mechanics for the active site, while including the influential environmental effects using the computationally less expensive MM.

Few enzyme systems have been studied with this method, and much work remains to improve the protocol, parameterization, and reliability of the technique. Thus the aim of this research is to develop protocols and simulation conditions for hybrid QM/MM calculations of LDH and MDH in an effort to determine the enzymatic reaction mechanism.




Rebecca Schmidt

Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research



v53 - VPP, PC



What are the results to date and the future of the work?

A large number of molecular dynamics (MD) calculations have been performed for the enzyme bound with either the inhibitor oxamate, the reactant pyruvate, or the product lactate. The purpose of these calculations is two-fold. Firstly, they provide relaxed starting structures for

- Appendix A


QM/MM studies. Secondly, it is more efficient to optimize the protocol for the classical region using these simulations which are less computationally costly than QM/MM calculations.

A variety of different simulation conditions were tested. The size of both protein and solvent were varied, and different force field parameters and protonation states of amino acids were tried. Results were compared to the crystallographic structure in terms of overall RMS deviations, occupancy of hydrogen bonds in the active site, torsions, and interatomic distances.

In general the hydrogen bonding patterns in the active site were reproduced in most simulations with distances between the substrate and key active site residues maintained accurately. Some difficulties arose, however, due to the flexibility of a mobile loop which closes over the active site. This mobile loop includes several amino acids which contact the substrate and are potentially critical for the reaction to occur, yet in many simulations this loop shifted considerably.

In certain simulations, however, the loop maintains its crystallographic orientation as well as important hydrogen bonds to the substrate. The parameters used in these simulations are consistent with the manner in which the force field was developed. The model system includes all protein residues within 24 Å of the active site as well as a 24 Å water cap, and the simulation employs a dielectric constant of e=1 with no counterions, neutralized amino acids, or harmonic constraints. Snapshots from these simulations are likely candidates for starting coordinates for future QM/MM calculations.

What computational techniques are used?

The Gaussian 94 package is used to perform ab initio quantum mechanical calculations. Amber 4.1 is used to perform standard molecular dynamics (MD) simulations and to analyze results. The QM/MM calculations will employ the locally developed MOPS program as well as the ChemShell package, currently being ported and modified as part of the Fujitsu Area 3 project.