Procedure

The common procedure has been to take the structures obtained with the reaction coordinate method as the input for the micro-iterative method. After the successful search a second derivatives analysis has been done to check the correct number of negative eigenvalues. Even though, there were always very small frequencies impossible to eliminate and probably caused by the typical coarse-grained surface inherent of enzymatic systems.

Obviously the method for the potential energy used here is the same used in section 2.3.

The options on the micro-iterative method described in the last section 3.2 are the following:

Core size

The core size has been selected in particular for every reaction. For most of proton transfer reactions where few atoms are expected to rearrange the core size is smaller than the quantum part. A good strategy is that when the search becomes difficult we can select a very small core (e.g. donor, acceptor and transferring proton) and perform, keeping the rest frozen, a fast TS search on this small system. Then this will be a very good initial structure for the micro-iterative refinement.

For the carbon configuration inversion the core includes all the residues that have QM atoms (as we already said, this reaction needs a bigger core size).

Frequency of both process

We always recommend a full TS search in the core before a new minimization of environment is restarted.

Interaction between the two zones

Since these PM3/AMBER calculations are not very expensive we do not select any of the approximations described in section 3.2. If the QM level became more expensive the QM(1SCF)/MM would be the most suitable strategy. Moreover, a full QM/MM evaluation at every minimization step permits the inclusion of some quantum atoms in the environment zone, in other words, a more flexible core size selection.