Hybrid Quantum Mechanical and Molecular Mechanical Studies of the Reaction Mechanism of Dihydrofolate Reductase

Principal Investigator

Jill Gready

Division of Biochemistry and Molecular Biology,

John Curtin School of Medical Research

The enzyme Dihydrofolate Reductase (DHFR) catalyzes the reduction of dihydrofolate to
tetrahydrofolate in the presence of NADPH co-factor. Tetrahydrofolate is important in the formation of DNA and thus DHFR is an attractive target for developing cytotoxic drugs to treat proliferative diseases such as cancer. As a first step, prior to a full QM/MM study of details of the mechanism, we have applied ab initio electronic structure techniques to determine the importance of the bulk enzyme environment in polarisation of the substrate and co-factor.
We aim to determine the magnitude of the polarisation, and what effect it might have on the reaction mechanism. We intend to investigate what features allow DHFRs from different organisms to perform the same biological function. A number of factors are being considered, including the importance of using realistic basis sets (incorporating diffuse functions), the sensitivity of the polarisation to different representations of the enzyme environment, the effect of different enzyme structures, and the effect of varying pH and the inclusion of water.



Stephen Greatbanks

Peter Cummins

Division of Biochemistry and Molecular Biology

John Curtin School of Medical Research

Alistair Rendell




w05 - VPP, PC



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

The calculations performed so far, using Hartree-Fock theory and representing the enzyme as point charges, have shown that the enzyme environment provides only a small degree of polarisation to the substrate and co-factor (measured in terms of changes in atom-centered charges and electron density). Particularly important is the fact that the polarisation experienced by the substrate and co-factor does not have the long-range, dipolar character shown in an earlier study in the literature, which was suggested to have major implications for the reaction mechanism. Calculations have also been carried out using the linear-scaling semiempirical code Mozyme, which allows the whole enzyme to be represented explicitly. Results from these calculations are in line with the Hartree-Fock results, in terms of the magnitude of the changes in charge distribution and the lack of dipolar polarisation.

What computational techniques are used?

The project has made extensive use of the Gaussian 94 program suite, requiring modifications to the code to increase the degree of vectorisation in link 502 (scf energy evaluation) to allow

- Appendix A

for calculations on these very large systems, near the limit of traditional Hartree-Fock techniques. The largest calculations are of ~1850 basis functions. Using the modified l502, speedups for the systems considered are more than a factor of 3. The calculations have required both large amounts of memory and substantial disk space. Modifications were also made to link 9999 to provide a selective checkpointing facility to reduce substantially the size of the restart files.
Appendix A -