INTtetr evolves towards the product overcoming the second FE barrier, that is only ≈ 2 kcal/mol high. At this second FE maximum, the new bond between the O𝛾 atom of S160 and the C5 atom of PEF4 is established and the ether bond between C5 and O5' is stretched, but not yet completely broken. Beyond this second energy hill, INTac-enz is formed, with the complete cleavage of the C5 -O5' bond (d2 = 3.24 ± 0.30 Å) and the protonation of the O5' atom (d6 = 0.98 ± 0.03 Å) by H237 which, in turn, slightly displaced from D206 (d9 = 1.98 ± 0.19 Å). This process yields the first leaving molecule of the catalytic cycle (PEF2'), with an associated reaction energy of ≈ 2.5 kcal/mol, thus resulting in less endergonic than the acylation of PET4.