FIG. 5.

Proposed five-step mechanism for the AgDI reaction. Step 1 leads to the formation of the first tetrahedral carbon center intermediate as a consequence of the attack by the activated thiol of Cys357. Asp96 and the nonprotonated primary N of the guanidinium group may induce deprotonation of the thiol group. A proton is extracted by His218, which forms a charge relay system with Glu157. In step 2 the tetrahedral intermediate collapses to the triagonal amidino intermediate, with liberation of ammonia. Asp96, Asp220, and His218 help stabilize the leaving ammonia and the positive charge development in the amidino group. In step 3 ammonia is replaced by water positioned for attack on the carbon center, interacting with the same groups as the ammonia. The intermediate revealed here by X-ray crystallography corresponds to one of the two complexes (either the ammonia or the water complex) with the amidino intermediate. Step 4 is the formation of the second tetrahedral carbon intermediate. His218 helps this step by abstracting one proton from water. The final step is the collapse of the tetrahedral intermediate to carbamoylputrescine and the regenerated thiol group.