However, when the 6-1R-hydroxyethyl group is restrained to position II, the barrier for the mutant enzyme drops to 15.7 kcal/mol. A further reduction (to 14.9 kcal/mol) is observed when a hydrogen bond between Asn132-ND2 and the 6α-1R-hydroxyl is enforced. For the TEM-1 Asn132Gly mutant, when the 6-1R-hydroxyethyl group is restrained to Position I (as observed in the wildtype) the barrier is only slightly higher than in the wild type (17.8 vs. 17.1 kcal/mol). However, the absence of Asn132 lowers the activation energy barrier to 16.4 kcal/mol when the 6αhydroxyethyl group dihedral is unrestrained and can exchange conformations between Positions I and III. Notably, the barrier increases significantly for TEM-1 Asn132Gly (to 25.2 kcal/mol) when the dihedral is restrained to Position II.