Three extreme possibilities have been proposed for the mechanism of NPP-catalyzed phosphoryl transfer. They are distinguished by the sequence in which bonds to phosphorus are made and broken. Though this phenomenon is subtle, it is important for understanding the physiological roles of AP superfamily enzymes, and also to molecular dynamic modeling.
1) A two-step "dissociative" (elimination-addition or D + A) mechanism that proceeds via a trigonal metaphosphate intermediate. This mechanism is represented by the red dashed lines in the figure at right.
2) A two-step "associative" (addition-elimination or A + D) mechanism that proceeds via a pentavalent phosphorane intermediate. This is represented by the blue dashed lines in the figure at right.
3) A one-step fully synchronous mechanism analogous to S2 substitution. Bond formation and breakage occur simultaneously and at the same rate. This is represented by the black dashed line in the figure at right.
The above three cases represent archetypes for the reaction mechanism, and the actual mechanism probably falls somewhere in between them. The red and blue dotted lines in Fig. 2a represent more realistic "concerted" mechanisms in which addition and elimination overlap, but are not fully synchronous. The difference in initial rates of the two steps implies different charge distribution in the transition state (TS).
When the addition step occurs more quickly than elimination (an AD mechanism), more positive charge develops on the nucleophile, and the transition state is said to be "tight." Conversely, if elimination occurs more quickly than addition (DA), the transition state is considered "loose."
López-Canut et al. modeled substitution of a phosphodiester substrate using a hybrid quantum mechanics/molecular mechanics model. Notably, the model predicted an AD concerted mechanism in aqueous solution, but a DA mechanism in the active site of Xac NPP.