Ghosh et al. (2013) in their article reporting the 4KJG structure said that the dephosphorylating mechanism of intestinal alkaline phosphatase was relatively well understood, and that it is known that the three bound metals are important although the role of the third one (which they called site M3) was not fully clear. The authors said: "The overall catalytic mechanism is well conserved between species. One Zn2+ ion (M1) activates the catalytic serine leading to the formation of phosphoserine intermediate. A water molecule activated by the second Zn2+ ion (M2) hydrolyzes the phosphoserine intermediate. The phosphate then gets released or transferred to an acceptor...."

Shown below is a partial image of the active site of 4KJG aligned with another member of the alkaline phosphatase family. 4KJG residues are in green, placental alkaline phosphatase residues are in yellow, and red dots represent water. The metal spheres also appear doubled because they are slightly offset in the two different 3D structures:

In the 4KJG intestinal alkaline phosphatase M1 and M2 are Zn2+ while M3 is Mg2+ . In other alkaline phosphatases such as the placental phosphatase there may be differences in the specific identities of the metals at these positions, and it can also vary between species. (There is also sometimes uncertainty about which specific metal it is, depending on the resolution of the structure.)

As noted when you were working with the 4KJG structure, the authors provided the enzyme with substrate (a phosphorylated form of 4NP) and then were able to obtain crystal structures with the enzyme bound to a product, 4NP
Links to an external site.
as shown above.

Think about the chemistry of the metal cations and the description of the mechanism (as well as previous discussion of the catalytic triad, and also review serine's structure here
Links to an external site.
if needed. Also keep in mind that the M1 metal may still be able to affect Ser92 even if it doesn't look very close in the image above! (In the image above it has already released product, so its conformation has somewhat shifted.)

The mechanism seems to rely twice on metals creating negatively-charged species... twice! You may recall that chymotrypsin also involved two sequentially-generated negatively-charged attackers (although metals were not involved).

Which two parts of the reaction mechanism sound like they become negatively-charged (i.e. nucleophilic attackers)?

Question

Ghosh et al. (2013) in their article reporting the 4KJG structure said that the dephosphorylating mechanism of intestinal alkaline phosphatase was relatively well understood, and that it is known that the three bound metals are important although the role of the third one (which they called site M3) was not fully clear. The authors said: "The overall catalytic mechanism is well conserved between species. One Zn2+ ion (M1) activates the catalytic serine leading to the formation of phosphoserine intermediate. A water molecule activated by the second Zn2+ ion (M2) hydrolyzes the phosphoserine intermediate. The phosphate then gets released or transferred to an acceptor...."

Shown below is a partial image of the active site of 4KJG aligned with another member of the alkaline phosphatase family. 4KJG residues are in green, placental alkaline phosphatase residues are in yellow, and red dots represent water. The metal spheres also appear doubled because they are slightly offset in the two different 3D structures:

In the 4KJG intestinal alkaline phosphatase M1 and M2 are Zn2+ while M3 is Mg2+ . In other alkaline phosphatases such as the placental phosphatase there may be differences in the specific identities of the metals at these positions, and it can also vary between species. (There is also sometimes uncertainty about which specific metal it is, depending on the resolution of the structure.)

As noted when you were working with the 4KJG structure, the authors provided the enzyme with substrate (a phosphorylated form of 4NP) and then were able to obtain crystal structures with the enzyme bound to a product, 4NP
Links to an external site.
 as shown above.

Think about the chemistry of the metal cations and the description of the mechanism (as well as previous discussion of the catalytic triad, and also review serine's structure here
Links to an external site.
 if needed. Also keep in mind that the M1 metal may still be able to affect Ser92 even if it doesn't look very close in the image above! (In the image above it has already released product, so its conformation has somewhat shifted.)

The mechanism seems to rely twice on metals creating negatively-charged species... twice! You may recall that chymotrypsin also involved two sequentially-generated negatively-charged attackers (although metals were not involved).

Which two parts of the reaction mechanism sound like they become negatively-charged (i.e. nucleophilic attackers)?

BlackTom AI · Accepted Answer

As we evaluate the mechanism, let's identify which components act as nucleophiles during the two key steps of catalysis.
Option 1: '4NP and M3' — 4NP is simply the substrate/product involved in monitoring turnover, not a nucleophile, and M3 is a metal ion (Mg2+ in some enzymes) that helps position or polarize substrates but does not itself provide a nucleophilic attack. This pairing doesn't describe the actual nucleophilic participants.
Option 2: 'Ser92 and a deprotonated water (i.e. OH-)' — Ser92 is the catalytic serine that forms the covalent phosphoserine intermediate, acting as a nucleophile after being activated by the surrounding residues and metal ions. The second attack step uses a water molecule that is activated (deprotonated) by the second metal ion (M2) to attack the phosphoserine, generating the inorganic phosphate. Thus, this option correctly pairs the two nucleophilic attackers in the mechanism: the serine and a hydroxide ion derived from water.
Option 3: 'His320 and Asp42' — These residues may participate in substrate binding or catalysis in other contexts, but they are not described as the primary nucleophiles in this mechanism. They do not form the key negatively charged attackers that attack the phosphate intermediate.
Option 4: 'M1 and M2' — While M1 and M2 are essential metals that activate the serine and water, respectively, they themselves do not act as nucleophiles attacking the phosphoserine; their roles are to polarize and activate substrates rather than to donate electron density as nucleophiles.
Option 5: 'Ser92 and a magnesium' — Although Ser92 is a crucial nucleophile, the second participant is not simply 'a magnesium' as a free nucleophile; the nucleophile is the deprotonated water activated by M2, not Mg2+ itself. This makes the pairing incomplete and misleading as a description of the actual two nucleophilic attackers.

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