TABLE 3.

The effect of constraints of different magnitude on the relaxation of the “deformed” water molecules of Structures 1 and 6 of Table 2 (see main text) relative to either the C=O or C-O⁻ species, after 1000 steps of QM/MM minimization using the MOLARIS simulation package³⁹ interfaced with Gaussian 03⁴⁰, with the MPW1PW91 functional⁴¹ and the 6-311++G** basis set.^[a]

Constraint	Structure 1		Structure 6
	Ketone (C=O)	Oxyanion (C-O−)	Ketone (C=O)	Oxyanion (C-O−)
Unminimized	15.1	−8.4	15.4	−14.2
5000	12.5	−10.0	12.9	−15.0
500	7.3	−17.2	8.4	−19.5
50	−1.0	−28.7	−3.8	−33.3
5	−11.1	−42.3	−13.5	−44.1
0.5	−14.8	−46.2	−15.2	−45.9
0.0	−15.2	−46.7	−15.4	−46.1

^[a]For comparison, the energy of the deformed structure with no minimization is also presented. All energies are given relative to the having the two water molecules at 5Å separation from either the C=O or C-O⁻ (as relevant), in kcal/mol (see Table S1 for the absolute energies of these structures), and the constraint is given in kcal mol⁻¹ Å⁻². From this table, it can be seen that in order to maintain the deformation, extremely large force constants of in excess of 50 kcal mol⁻¹ Å⁻² are required, and that the smaller constraints result in a relaxation to a more optimal structure. Note that a typical force constant in a protein for a small deformation is, at most, 5 kcal mol⁻¹ Å⁻², and thus it is not possible to maintain the deformation without an unphysically large force constant. This indicates that using large constraints may lead to completely unrealistic results.