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


Principal Investigator

Jill E. Gready

John Curtin School of Medical Research


Rebecca K. Schmidt

John Curtin School of Medical Research


v53 - VPP, PC, MDSS

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 with 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.
A variety of computational techniques are employed in this project to study these enzymes. In particular, the relatively new hybrid quantum mechanical and molecular mechanical (QM/MM) technique is used to characterize the chemical reaction. 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 main 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.

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

In the hybrid QM/MM method, 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 (MM). The optimization of the protocol and parameters for the classically treated protein environment has been completed this year after the analysis of many different MD simulations. These simulations reveal that it is important to model correctly the placement of a mobile loop which closes over the active site. The position of this loop is sensitive to how much of the full tetrameric system is included in the simulations as well as the balance of long-range nonbonded interactions. A set of simulation conditions has been identified which result

Appendix A -



in trajectories in which the position of the mobile loop is physically realistic, and this protocol was used to generate starting structures for QM/MM studies.

A quantum mechanical study of the conformational flexibility of the substrates (pyruvate and lactate) and inhibitor (oxamate) of LDH was also completed this year. This study indicated that the semiempirical method which will be employed in the hybrid QM/MM calculations (AM1) adequately models these molecules. Now that these two studies have been completed, hybrid QM/MM calculations will be started.

The MDSS was essential for this project, as large amounts of data were generated by the numerous MD simulations. The trajectories could be accessed easily and quickly when further analysis was required.

What computational techniques are used?

The GAUSSIAN94 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.








- Appendix A