P. Berglund et al. / Tetrahedron: Asymmetry 10 (1999) 4191–4202
4201
to relax in the force field: all enzyme hydrogens were allowed to move during a dynamics simulation of
1000 fs length, and the energy for the hydrogens in the last structure was minimized. The above steps
were repeated for the water hydrogens. Then the energy for all hydrogens was minimized and finally the
energy of all atoms was minimized. The inhibitor was then removed and replaced with a built substrate.
The substrates were assigned empirical charges29 and atom types consistent with the force field. The
substrate was allowed to relax in the enzyme through repeated dynamics simulations and minimizations
on subgroups of the substrates: first the hydrocarbon chain in the acyl part of the substrate was allowed
to move for 1 ps and the last structure was minimized. Then the phenoxy group, the alcohol group, and
finally the whole substrate one at a time were allowed to relax in the same way, whereafter, the energy of
the whole system was minimized. The same procedure was repeated for two different conformations of
the two enantiomers for three substrates, giving a total of 12 structures. These 12 minimized structures
were the starting points for dynamics simulations, lasting for 100 ps each. In the dynamics simulations
all atoms were allowed to move. All bond lengths were set to constant values using SHAKE,30 which
made it possible to use a time step of 2 fs. The structure was heated with 50 K per 10 ps up to 300 K,
giving a total heating time of 50 ps. All modeling was performed with the software package SYBYL6.5
(Sybyl6.5, Tripos Inc., 1699 South Hanley Rd., St Louis, Missouri, 63144, USA) on an SGI Octane
computer. Tripos’ implementation of the Amber force field31,32 considering all atoms was used.
Acknowledgements
Financial support from the Swedish Council for Forestry and Agricultural Research (SJFR) and
from the Swedish National Board for Industrial and Technical Development (NUTEK) is gratefully
acknowledged.
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