The Arg166 likely interacts with the following oxygens in the active site:

a) Oxygen 1 in S1 b) Oxygen 2 in S1 c) Oxygen 3 in S1 d) Oxygen 4 in S1

e) Oxygen 5 in S1 f) Oxygen 6 in S1 g) a and b h) c,d, and f i) c and e

The above is a proposed mechanism adapted from K.M. holtz, E.R. Kantrowitz| FEBS Letters 462 (1999) 7-11. The active site of the Alkaline Phosphatase (Step 1) forms an enzyme-substrate complex (Step 2) by coordinating the ester oxygen of the substrate to Zn1 and additional interactions with the non-bridging oxygens of the substrate with Zn2 and the Arginine 166 amino acid. The ROH then leaves and an enzyme-phosphate intermediate is formed with the phosphate covalently bound to the enzyme while interacting with the Zincs and Arginine in the Active Site. A nucleophilic hydroxide coordinated to Zn1 then attacks the phosphate and forms a non-covalent enzyme-phosphate complex (Step4). The phosphate may then leave. In this diagram, the Mg2+ and other amino acids involved with interaction to the Zinc are not shown. enzyme-phosphate complex (Step4). The phosphate may then leave. In this diagram, the Mg2+ and other amino acids involved with interaction to the Zinc are not shown. Alkaline Phosphatase 8O9O