Furthermore, we have assessed mutations of the tyrosines involved in the proton relay using Y115F and Y467F mutants (Figures and). These variants exhibited decreased specific activities of 32 and 58% with respect to the WT enzyme. These mutations practically did not change the height of the TS1 barrier (differences within 1 kcal/mol) but lowered the stabilization of E:I by 2.6 kcal/mol and slightly increased the energy of TS2 (1.1 -3.4 kcal/mol). This effect may be associated with the decreased polarity of the active site upon substitution of the tyrosine residue by phenylalanine and with partial disruption of the proton relay system, which utilizes both Y115 and Y467 (Figure ).