QM/MM calculations were carried out with QSite. The topological analysis of the electron density, involved in the AIM approach, is built into the QSite code and is invoked with iplotnoncov=1. Natural Bond Orbital calculations were done with NBO 6.0 which is implemented in Jaguar 10.1. The X-Ray crystal structure, 1IDQ, was preprocessed with Schrodinger Protein Preparation Wizard. The Schrodinger Protein Preparation Wizard may flip the orientation of Gln, His and Asn residues in the enzyme structure to optimize the hydrogen-bonding network because the orientation of these residues is unclear from the X-ray crystal structure. The titratable residues (i.e., histidines, aspartates, glutamates) outside of the QM region were protonated assuming pH 7.0 because the PROPKA method for calculating the pKa values of amino acid residues failed for this structure. The Protein Preparation Wizard detected that the following residues were incomplete in the X-ray crystal structure: Glu83, Glu119, Gln120, Pro121, Asn122, Pro123, Asn124, Pro125, Asn128, His185. Although they were on the surface of the enzyme and unlikely to affect the calculations, they were repaired. Furthermore, the hydroxyl hydrogen (H28) of Ser402 was rotated to point to the equatorial O3 of the vanadate. (See Figure Two QM regions were used. The first QM region, which is shown in Figure , consisted of His496 (side chain), Lys353 (side chain), Arg360 (full), Arg490 (full), Ser402 (full), Gly403 (full), HIE404 (full) [31], Asp292 (side chain), H2O1911, H2O1778 and vanadate (VO4) in various protonation states. We will refer to this first QM region as "QM Region I" (Figure ). The second QM region, "QM Region II" (Figure ), consisted of these 8 residues and vanadate as well as all of the atoms in the following residues: Ala399-Pro398-Phe397-Pro396-Pro395. It is also included two additional crystallographic water molecules: H2O1832, H2O1628. The series of three water molecules-namely, H2O1911, H2O1778, and H2O1832-form a hydrogen bonded bridge between the axial oxygen (O4) of vanadate and Asp292 (i.e., O13). QM Region I consisted of 149-153 atoms depending on the protonation state of vanadate. QM Region II consisted of 226-230 atoms.