The proposed covalent intermediate is formed through nucleophilic attack by the enzyme.
Red arrows indicate nucleophilic attack.
These threonines are both able to act as primary nucleophiles. They are both necessary for activity, and neither kinetic nor structural data has yet been able to show which is the most likely primary nucleophile. Click the image to enlarge. It has been modelled and refined with the aspartate ligand covalently bound to T12 through its side chain carboxy group, in a structure which may resemble an acyl-enzye intermediate. This indicates at least that T12 may act as the primary nucleophile, although this activity may be caused by the lack of a nucleophilic group on residue 89.